1961;21:193-197. Cancer Res   S. Dales and A. F. Howatson  Virus-like Particles in Association with L Strain Cells

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Virus-like Particles in Association with L Strain Cells

S. DALES*ANDA. F. HOWATSON

(Division of Biological Research, Ontario Cancer Institute, Toronto, Canada)

SUMMARYElectron microscope studies of three sublines of Earle's L strain cells showed that

in two of the three sublines virus-like particles of type C were present within cyto-plasmic vacuoles and outside the cells close to the cell surface. The particles formed bya budding process at the ceil membrane. Similar particles were observed in pelletsprepared from the medium in which the cells had been grown, and in cells of tumorsproduced in C3H mice by inoculation of the cultured cells. A second type of particle,type A, was observed within cytoplasmic vacuoles in all three sublines of cells cultivated in vitro. No biological activity has so far been associated with either type ofparticle.

Since methods for preparing cells and tissues forthin sectioning and electron microscopy have become available, considerable effort has been expended in searching for structures that might beidentified as viruses within and around cells ofmany different types of tumors. In tumors ofknown viral etiology these efforts have been forthe most part successful in that particles presumed to be the causative agents have been observed in many instances (3), although it shouldbe noted that in only a very small number of thesehas the further step required for unequivocalidentification been made, namely, the establishment of a correlation between the number of particles of the type observed and tumor-inducingactivity (25). In a number of tumors for whichthere is no biological evidence of viral origin, structures morphologically similar or identical to thoseobserved in certain virus-induced tumors havebeen described (3). The significance of such "virus-like" particles is difficult to assess in the absence of

any demonstrable biological activity. Nevertheless, it cannot be assumed that their presence inand around tumor cells is fortuitous, and it is ofsome importance to determine their relation to thetumor cells and frequency of occurrence in different tumors. This is especially true in view of current investigations of the possible viral etiology ofhuman tumors in which morphological observations may be important, since many experimental

* Present address: Department of Cytology, The Rockefeller Institute, New York 21, N.Y.

Received for publication July 22, 1960.

approaches used with animals are not feasible inhuman beings.

In the present report two types of virus-like(VL) particles that have been observed in association with certain lines of Earle's L strain cells are

described, and their significance is discussed. Theorigin and relative malignancy of the cell linesused are described in the following section.

MATERIALS AND METHODSDerivation of cell lines.—Earle's strain L cells

were originally derived in 1943 from an expiantof subcutaneous connective tissue which had beentreated in vitro for several months with methyl-cholanthrene (8). Upon injection into adult C3Hmice these cells were found to be highly malignant,producing sarcomas in 68 per cent of the mice injected. After repeated passage of the cells in vitrothis high incidence of tumors declined to only 1 percent. In 1948 L cells were cloned by Sanford et al.(20), and a permanent line of cells, the NCTN 929line, was established; this was found to producetumors in a small percentage of adult C3H mice.However, the tumor incidence in adults could beincreased from 1 to 65 per cent by irradiating thehosts before injection (9), and more recently it wasshown that the incidence of tumors produced byinjecting newborn mice was very high (19). Theseresults showed that the NCTN line of L cells wasstill highly malignant, although the adult animalshad apparently acquired some immunity againstthese cells. Several sublines of the clone NCTN929 have been developed, three of which were used

193

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194 Cancer Research Vol. 21, February 1961

in the present investigation. These sublines havebeen designated L2, AMK2_2, and L4CZ. The derivation and characterization of the first two of thesesublines of L cells have been described in detail(19). Briefly, the 1,2 cells were obtained from astock of frozen L cells which had been kept at—70°C. for several months; the AMK2_2 cells

were derived from a clone obtained by dilution andplating of the AMK subline of cells (which iskaryologically almost identical with the L2 sub-line); L4CZ cells were recently derived from L2cells by Axelrad and Evans1 in the following way.Suspensions of L2 cells were injected into newbornC3H/HefOci mice. Cells of a tumor which grewfrom these injected cells were then re-injected into2-3-month-old mice of the same strain. A secondtransplant tumor arising in one of these animalswas again transplanted. After dispersal by tryp-sinization, the cells of this tumor grew in suspension in the same way as the L2 and AMK sublines.Although karyotypically L4CZ cells are very similar to the L2 cells, they are much more malignant,having retained their high malignancy after manygenerations of culturing in vitro and several passages in vivo. About 44 per cent of C3H mice giveninjections at birth of IO2 L4CZ cells develop tumors, whereas only 4 per cent of the mice whichreceive a similar dose of L2 cells develop malignantgrowths.

Specimen material.—Electron microscopestudies were made on three types of specimen: (a) cells of each of the three sublines cultivated m vitro, (b) tumors that resulted from giving injections to newborn C3H mice of cells of thethree sublines, and (c) pellets prepared from thenutrient media in which the cells had been grown.

The cells were propagated in vitro in suspensionculture by the technic of Siminovitch et al. (22),the nutrient medium being CMRL 1066 (11)supplemented with 10 per cent horse serum. Thesecells and also the tumors that arose from them inmice were prepared for thin sectioning and electron microscopy by standard procedures. The pellets were prepared in the following way. About1-2 X IO7 cells, sedimented by low-speed cen-trifugation, were suspended in 30-50 ml. of freshnutrient medium and were incubated in suspension for approximately 24 hours. On the followingday the cells were again sedimented and were oncemore suspended in fresh medium and incubated.The supernatant fluids obtained after the removalof cells following each 24-hour period of culturingwere processed separately. Each sample of fluidwas centrifuged at an intermediate speed(20,000 X g for 15 minutes) to remove some of

1Unpublished experiments.

the cell debris. The supernatants were then centrifuged 104,000 X g tor 60 minutes. The pelletsformed were resuspended in 20 ml. of saline solution with 0.125 per cent trypsin added and wereincubated for 30-60 minutes at 37°C. to digest

some of the remaining protein material. Pelletsprepared by a final centrifugation at 104,000 X gfor 60 minutes were processed for electron microscopy in the same way as the cells and tumor tissue. To enhance the contrast of thinly sectionedspecimens two procedures were employed. In thefirst, specimens were "stained" for 20 minutes

with a 1 per cent solution of uranyl acetate dissolved in 70 per cent alcohol prior to further dehydration and embedding. In the second, thinsections were "stained" with lead hydroxide by

the procedure of Watson (24).

RESULTSThe components of fine structure generally ob

served in cells of the fibroblast type were presentin Earle's L cells. Thus, in the cytoplasm of thinly

sectioned cells there were numerous oval andelongated mitochondria, many smooth- and rough-walled vesicles of varying size, ribonucleoproteingranules dispersed throughout the cytoplasm, andfine processes emanating from the cell membrane.Examination in the electron microscope of thinlysectioned L cells of the L2 and AMK sublines,which had been cultured in vitro, revealed in addition to normal components the presence of virus-like (VL) particles attached or close to the cellmembrane. These occurred in 10-20 per cent of thecells examined, usually singly or in small groups;but occasionally larger aggregates were observed,such as the one illustrated in Figure 1. Most ofthese particles were spherical and ranged in sizefrom 80 to 110 m/j, but a few were as large as 180m p in diameter. Generally, each particle containeda centrally placed nucleoid surrounded by a lessdense zone and a limiting membrane, but occasionally particles devoid of dense centers were alsoobserved. The nucleoid was variable in density andappearance, sometimes being uniformly dense butmore often consisting of a dense rim with a lessdense, usually filamentous interior. This variability might be due to the presence of incompleteforms of these particles or could result from differences in the quality of preservation and staining.Similar particles were less frequently observedeither singly or in groups within membrane-boundcytoplasmic vesicles. Figure 2 shows an area ofcytoplasm in which there are a number of vesicles,two of which contain groups of VL particles. Mostof these particles fit the morphological category"type C," proposed by Bernhard (6). Protuber-

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DALESANDHOWATSON—Virus-likeParticles icith L Strain Cells 195

anees of the cell membrane or the membrane surrounding cytoplasmic vacuoles were commonly observed at, or close to, the sites where VL particleswere present. The protuberances were limited onthe outside by a membrane that in many instancesappeared to be continuous with the cell membrane.On the inner surface there was an accumulation ofdense amorphous material and a second very densemembrane. In Figures 3 and 4 are shown what areinterpreted as stages in the formation of VL particles; the protuberance in Figure 3 (right) represents an early stage; in Figure 4 the "budding"

process is more advanced, and in Figure 3 (left)is shown a particle apparently almost separatedfrom the cell. This particle differs from typicaltype C particles in that the center is less dense andthe surrounding zone denser. Particles similar tothe one illustrated were found among type C particles close to the cell membrane and also withincytoplasmic vacuoles. They may represent freeparticles which are incompletely developed. It islikely, however, that some of these representprotuberances that have been sectioned parallelto the cell surface.

Occasionally finger-like projections much longerthan the protuberances already described were observed at the cell surface at locations where VLparticles were present or forming. These were of adiameter approximately the same as that of theVL particles. One ot these projections is shown inFigure 5. Apart from its greater length (approximately 400 mju) it is similar in structure to themuch commoner short protuberances, suggestingthat it is closely related to them.

In addition to the particles and projections fromthe membrane that were present in cells of the L2and AMK sublines, there were in all three sublinescultured in vitro smaller particles of a differentmorphology. These were usually distributed singlywithin cytoplasmic vesicles. They measured 60-80mu in diameter and had a center of low densitysurrounded by a double-layered membrane. Figure 7 shows a portion of the cytoplasm of an L2cell in which there are cytopiasmic vacuoles containing particles of the second type, indicated byarrows. These particles are of type A, accordingto Bernhard's classification (3). In the same fig

ure, near the surface of the cell, are shown a fewparticles of the first type described, type C.

Many of the type C particles appeared to beseparated from the cells. To test whether theywere released into the nutrient medium, supernatant fluid from ceil cultures was centrifugedinto pellets, as previously described. Examination of thinly sectioned pellets in the electronmicroscope routinely showed the presence of

large numbers of particles identical to the type Cparticles seen in sectioned cells. A typical area ofa sectioned pellet is shown in Figure 8. A smallgroup of VL particles is shown in Figure 6 at highermagnification. These type C particles were seenonly in pellets prepared from supernatant fluidsobtained from cultures of L2 and AMK cells. Notype A particles were seen in these sectioned pellets. Neither type A nor type C particles werefound in pellets of supernatant fluids preparedfrom L4CZ cultures, nor in pellets prepared fromfresh nutrient medium.

TABLE1SUMMARYOFOBSERVATIONSONTHE

OCCURRENCEOFVLPARTICLES

MATERIALEXAMINEDSolid

tumors produced bysublines AMK andLjSolid

tumors produced bylineL4CZCells

of sublines AMK andLI cultured insuspensionCells

of subline L4CZ cultured insuspensionPellets

from nutrient mediumin which L2and AMK cellshad been grown for 24

hoursPellets

from nutrient mediumin which L4CZ cells hadbeen grown for 24hoursPellets

from fresh nutrientmediumLOCATION

ANDTTPE orPARTICLESType

ACytoplasmic

vacuolesCytoplasmic

vacuolesCytoplasmic

vacuolesCytoplasmic

vacuolesNot

foundNot

foundNot

foundTyp«

CCytoplasmic

vacuoles cellmembraneNot

foundCytoplasmic

vacuoles cellmembraneNot

foundPresentNot

foundNot

found

Examination of the tumors grown in C3H miceshowed that particles of type C, identical in appearance to those found in cells cultured in vitro,were present in tumors induced by L2 and AMKcells but were absent in tumors induced by theliiCZ subline. However, tumors induced by allthree sublines of cells contained type A particles.The observations on sectioned cells and pelletshave been summarized in Table 1.

DISCUSSIONWe shall consider first the type C particles.

These particles have the general morphologicalcharacteristics that are associated with manyknown viruses including several tumor viruses,

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196 Cancer Research Vol. 21, February 1961

namely, a dense central body or nucleoid surrounded by a less dense zone, the whole being envelopedin a dense limiting membrane. It is possible thatthey represent the etiological agent that was responsible for the original conversion of the cells tomalignancy, but there is no evidence in support ofthis; and it would seem unlikely, since one sublinedid not contain any particles. Nevertheless theparticles may represent tumor virus. There arenow several instances of the recovery from tumortissue of tumor virus of a type apparently unrelated to the agent causing the tumor from whichit was recovered. FOPexample, polyoma virus hasbeen recovered from leukemic tissue (23) and frommammary tumor (16) of the mouse in the induction of which other viruses, both distinct frompolyoma, are implicated. Again, a virus causingleukemia has been obtained from a sarcoma forwhich there is no evidence of viral origin (17).Thus it would seem that tumor viruses may becarried as passenger viruses and that tumor cellsprovide a suitable environment for them. Further,the mode of formation of the particles that we observed around L cells, namely, a budding processat the cell membrane, closely resembles that described for certain tumor viruses, the identity ofwhich is reasonably well established (3). The mostdetailed studies have been made by deHarven (10)on the leukemia virus of Friend. Thus, from apurely morphological standpoint, the VL particlesare indistinguishable from known tumor virusesboth in structure and in mode of formation. However, as already pointed out, the significance ofsuch morphological evidence is open to questionin the absence of demonstrable biological activity.

To test whether the particles released from thetwo strains of L cells had any biological, particularly tumor-inducing, activity, pellets of the particles obtained as described were resuspended inbuffered saline solution and injected subcutane-ously into newborn CSHmice and Syrian hamsters.To date, 8 months later, no biological activity ofthese preparations has been detected. These negative results do not, of course, rule out the possibility that the VL particles are in fact tumor viruses, for it is known that the action of sometumor viruses is strongly conditioned by host factors, and the conditions required to demonstratebiological activity may not have been satisfied inthe limited tests that we have carried out.

Another possibility is that the VL particles area nontumor passenger virus. Of nontumor viruses,the myxoviruses probably bear the closest resemblance to the VL particles in size, appearance,and mode of formation (2, 18). However, injection of suspensions of VL particles into mice pro

duced none of the symptoms associated withmyxoviruses, and inoculation onto cultures ofmouse embryo cells had no apparent effect. Further, the presence of the VL particles in no wayaffected the susceptibility of L cells to infectionby vesicular stomatitis virus, whereas L cells infected with myxoviruses are not susceptible whensubsequently exposed to vesicular stomatitis virus(12). Thus, there is no evidence that the VL particles are related to the myxovirus group. Attempts to induce the production of VL particlesin the ÕJtCZline by inoculating cultures of thesecells with suspensions of particles obtained fromthe other two lines were not successful. Thus, allour attempts to associate biological activity withthe type C particles have failed, and there is noevidence other than their strong resemblance instructure and mode of formation to known virusesthat the particles are viral in nature.

A remaining possibility is that the particles arenot infectious units but are a product of some kindof secretory process in the cells. This presumedsecretory activity did not seem to disturb cellularfunction, for the lines of cells that produced particles grew as rapidly and appeared as healthy asthe one that did not. Further, particle productionwas not influenced by environmental factors, sincethe two lines that produced particles did so whether grown in vitro or in vivo.

The viral nature of the type A particles is alsoquestionable. It appears that they are not releasedfrom the cells as are the type C particles, becausethey were not present in the pellets prepared fromthe nutrient medium in which the cells weregrown. It was therefore not possible to test thetype A particles for biological activity in thesame way as the type C particles. Their claim tobe considered as possible viral agents rests onpurely morphological grounds. Particles of type Ahave been described in cells of many differenttypes of tumor in mice. They may occur sparselyor in large aggregates in tumors of known viraletiology such as mouse mammary tumors (4, 5),or they may be present in tumors for which viraletiology has not been demonstrated (15). It hasbeen suggested that they represent a stage in theformation of mature virus particles (3), but theymay equally well be a reaction of the cell to thepresence of virus or to some other abnormal condition. Although in some tumors a close associationhas been observed between the Golgi region,known to be concerned in secretory activities ofthe cell, and type A particles (5, 15), it is not evident in L cells. Nevertheless, the possibility thattype A particles in L cells represent some abnormaltype of secretion should not be overlooked. Type A

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Fio. 1.—Part of an Lj cell from a suspension culture. A largenumber of type C particles are shown in association with thecell membrane. X 44,000.

FIG. 2.—Portion of the nucleus (upper right) and cytoplasmof an LScell from a suspension culture, showing two groupsof type C particles within cytoplasmic vacuoles. X 44,000.

FIGS. 8-5.—These Figs, illustrate stages in the formationof type C particles at the cell membrane.

FIG. 8.—Within both the protuberance of the membraneand a circular structure which lies in close proximity to it,there is a dense inner membrane. The circular structure maybe an incompletely developed type C particle, or it may bethe cross section through another protuberance which wasoriented perpendicularly to the plane of section. X 128,00(1.

FIG. 4.—Amicrovillus with a VL particle forming at itstip. Note the dense material closely applied to the tip of themicrovillus and the very dense semicircular inner membrane.XI 28,000.

FIG. 5.—Afinger-like projection at the surface of an LÎcell. Apart from its greater length, its morphology is similarto that of the structures shown in Figs. 3 and 4. X 128,000.

FIG. 6.—Detail of type C particles, from a sectionedpellet similar to that shown at lower magnification in Fig.8. X200,000.

FIG. 7.—Area in the cytoplasm of an Lj cell illustratingtype A particles (indicated by arrows). Each has a doublelimiting membrane and a center of low density. At the upperright of the micrograph is a group of type C particles near thecell membrane. X38.000.

FIG. 8.—Area of a sectioned pellet of partially purifiedVL particles. X38.000.

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DALES AND HOWATSON—Virus-like Particles with L Strain Cells 197

particles seem to be associated with neoplasia, forwhich they have not to our knowledge been described in non-neoplastic cells; but their role andsignificance, if any, in .the neoplastic process remains to be determined.

There is now a long list of tumors both in animals and in human beings in association withwhich particles suspected of being virus have beenobserved, but for which no viral activity has beendemonstrated biologically. These include Ehrlichascites tumor cells (1, 21), plasma-cell tumors (15),human leukemic lymph nodes (7), and mammarytumor (14), and uterine epithelioma (13) in the rat.Whereas such observations are of interest andmay be of great significance, it is clear that moreevidence must be acquired before any claim to arole in carcinogenesis can be attributed to particlesbecause of their resemblance to known viruses.The present studies show that, even when it ispossible to isolate the "virus-like" particles in

fair quantity and in fairly pure state, it may stillbe difficult to obtain evidence that they have anybiological activity.

ACKNOWLEDGMENTSTins investigation was carried out during the tenure by one

of the authors (S. Dales) of a post-doctoral fellowship of theNational Cancer Institute of Canada. It was supported bygrants from the National Cancer Institute of Canada and theFoster Bequest Fund of the University of Toronto. The authors wish to thank Dr. A. A. Axelrad for providing one of thecell lines used in the investigation.

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