The histological changes following inoculationof the RPL-12 lymphoma into the pectoral muscleand brain of the chicken have been described (29,30). Superimposed infection of the host with theN.F.T. strain of St. Louis encephalitis virus results in the development of prominent cytoplasmic“inclusions―in the tumor cells during the periodwhen the virus concentration in the tumor is highest (30). Apart from some displacement or indentation of the nucleus by the “inclusions―and occasional margination of the chromatin, there isno further morphological evidence of degenerationbefore the tumor cells in the pectoral muscleundergo phagocytosis. When the virus is inoculated into the host with tumor growing in thebrain, phagocytosis is deficient, and an abnormallylarge number of metaphase mitotic figures areobserved in the tumor cells containing “inclusions― (30). The present experiments were designed to analyze the formation and structureof the so-called “inclusions,―and to determinethe effect of virus infection on the mitotic processby comparing the cytological and cytochemicalproperties of the tumor cell before and after infection with virus.

MATERIALS AND METHODSComplete details of the biological procedures

and the plan of the experiments have already beenpublished (29, 30).

Tisauea.—k addition to the routine histological examination of all organs and tissues, the following material wasspecially prepared for cytological and cytocbemical study:(a)pectoralmuscleandbraintissuecontainingtumor,dailyfrom the time of inoculation of virus until tumor cells in themuscle had undergone phagocytosis, or until the birds withtumor in the brain had died; (6) uninfected tumor tissue on thesame days as (a).

Preparation of the iiaauea.—Thin fragments were scrapedwith a sharp knife from the cut surface of the tissue andsmeared on slides. When no fixative was used (Table 1), the

Received for publication June 18, 1954.

smears were allowed to dry for 5 minutes before staining;when fixation was desired, the slides were immersed in thefixative while still wet. Small pieces were placed in 0.85 percent NaCl or mixed with supravital staining solutions on slidesand examined directly by ordinary and phase microscopy.Blocks of tissue were fixed as described below, and paraffinand frozen sections were prepared. Except where otherwisestated (Table 1), the final preparations were mounted inPermount, and the paraffin-imbedded material which had beenfixed in formol sublimate was treated with iodine and thiosulfate before staining.

Fixation and .iaining procedure..—Paraffin sections ofmaterial fixed in formol sublimate were stained with Barrett'sstain (4) to observe the general cytological detail. For thedemonstration of Golgi bodies and mitochondria, blocks werefixed and prepared by standard methods: (a) Cajal's silverand the Sjovall and Kolatchew osmic acid methods for Golgibodies (88) and (6) Aitmaun's and Regaud's methods formitochondria (88). In addition, the more recent method ofHarman (22) was applied to blocks fixed in Regaud's fluid.Supravital staining properties were investigated by placingsmall fragments of tissue in freshly prepared mixtures ofstock solutions of 0.006 per cent Janus green and 0.01 percent neutral red in 0.85 per cent NaC1 in the proportions of2: 1, 1 :1, and 1:2. A comparison of the sudanophilic with thesupravitally stainable material in the cells was made by replacing the supravital stain under the cover slip with Baker'sSudan black B (1) and differentiating with 50 per cent alCOhOl.

To minimize repetition and clarify the rationale, the cytochemical procedures are outlined in Table 1.

Anolyair of mi@oais.—Owing to the rapid phagocytosis ofinfected tumor cells in the pectoral muscle, only brain materialwas available for sustained analysis of the mitotic process insections stained by the Feulgen method. Beginning on the daywhen inclusions were first observed, 1,000 tumor cells in eachsection were analyzed, the number of cells in the various phasesofmitosis was noted, together with the proportion of binucleatecells and the incidence of gross mitotic abnormalities as definedby Glucksniann (17). Multipolar division, bridge formation,

and lagging of an occasional chromosome in anaphase werenot regarded as abnormal for the purposes of the count, sincethey do not necessarily lead to death of the daughter cells.

RESULTSUNDirECTED TUMOR Csu@a

Characteriza!ion of the structures of the cell.—Supravital staining with neutral red and Janusgreen revealed numerous rounded green mitochondria of regular size distributed throughout

758

Studies on a Transplantable Chicken Tumor(RPL-12 Lymphoma)

III. Cytological Changes during Virus—inducedOncolysis

ROBERT LOVE AND GEORGE R. SHARPLESS

(Viral and Rickett.@alResearch, Lederle Laboratories D@viston,American Cyanamid Company, Pearl River, N.Y.)

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TABLE 1

CYT0cHEMIcAL PROCEDURES

MaTson or:Preparation Staining

Par. fros., and Sudan black B (1)smear Sudan Ill and 117(87)

Fros.and SudanblackB aftersmear lipid extraction

Par. Sudan black B

Frog. and Cain's Nile blue nilsmear fate (8)

Fros. and Baker's acid hemasmear tin (2)

Smear Schiff (84)

Smear Performic acid Schiff(25)

Frog.and Schultz(89)smear

Par. fros. and P.A.S. (84)smear

P.A.S. after acetylation in pyridine(25)

P.A.S. after lipid cxtraction

P.A.S. followed bySmear Sudan black B

P.A.S. after bromination

All material for lipid investigation, and for comparison with any lipid stain, mounted in glycerincjelly

Lipid extracted by 6 hours' immersionof slidesinhot chloroform-methanoL

To insure that sudanophiliawasnot due to chemical combination with Sudan black it was shownthat the dye, but not the lipid, could be removedby acetone (26).

Compared with preparations stained with Sudandyes to confirmthe lipid nature of the materialstained.

Direct Schiff for preformed aldehyde; plasmal foracetal phosphatides, and Schiff after exposure toair for S days for aldehyde secondary to oxidation of unsaturated fatty acids (20).

Myelin stained simultaneously as a positive controL

Mouse adrenal stained simultaneously as a positivecontroL

In all cases where P.A.S. stains were used, controlsections ofthe same material were treated identically except that oxidation with periodic acidwasomitted.

Control section stained after treatment with pyridine alone to determine reduction in P.A.S. staindue to solution of cell constituents in pyridine.

The lipid extraction process did not affect the intensity of the P.A.S. reaction of control sectionsof clam. A differencebetween the unextractedandtheextracted material must bedue to P.A.S.positive lipid.

To determine whether location of the lipid coincides with that of the P.A.S.-positive material.

Control section of clam5 stained before and afterbromination showed considerable reduction inthe intensity of the P.A.S. reaction. Brominationby exposure to bromine vapor in bright sunlightand by Lillie'smethod (25).

Activity of enzyme shown by its ability to reduceiuetaehromasia of clam acid mucopolysaccharides.5

P.A.S. reaction of control section of liver glycogenreduced by malt diastasepreparation used.

Counterstained with fast green FCF.Stained before and after incubation for 1 hour in

buffered ribonuclease (27); staining of controlsections tmaffectedby incubation in buffer alone.Association of stainable material with lipid constituents of the cell investigated by staining withtoluidine blue after lipid extraction. Fixation oftoluidine blue stain by the method of Bensley (5).

For amino acidsand ?purines and pyrimidines.

Forresidual stainingtherblocldngamino acids.

Destruction of RNA by strong acid for 16 hours(15), and of amino acid reacting groups by benzoylation.

To assess amount of stainable material removed bypyridinc.

Aniline blue, 0.01 gus.; orange G, 0.1 gm. in 100ml. 2per cent aceticacid.

Control smear negative after inactivation of enzyme with fluoride and incubation in substratefor 6 hours.

Control section of clam positive.5

Sinceresults werenegative, cellsfrom the Ehrlichascites tumor which were known to be positivewere run simultaneouslyas controls.

Ratadrenal stained simultaneously as positive controL

Puarosa: TOIzv@TIGATg

Lipid

Neutral and acidic lipid

Phospholipid

Fatty aldehydesand precursors

Unsaturated fattyacids

Cholesterol

P.AS.-positivematerial

1,2-Glycol groups

P.A.S.- positivelipid

P.A.S.-positivelipid

P.AS.-positive(unsaturated)lipid

Acid mucopolysaccharides

Glycogen

DNARNAAcid mucopoly

saccharides

Tyrosine, tryptophan,histidine,? purines andpyrimidines

Histone

Acid phosphatase

Alkaline phosphatase

Succinic dehydrogenase

FixationF. sub., f. calc.

and none

F. sub., f. caic.

F. sub.

F. sub. andf. caic.

F. calc.

None

F. sub., f. caic.and none

F. caic. andnone

F. sub., f. calc.and none

F. sub. andf. caic.

F. sub. andf. calc.

Smear

Fragmentsoftissue diracily insubstrate

F. sub. andf. cak

F. sub. andf. caic.

Lipidextraction, hyaluronidase(27),P.A.S.

Lipidextraction dinstase(@7), P.A.S.

Feulgen (87)MBE (87)0.01 per cent tolui

dine blue (5)

Coupled tetrazoniurn(10)

Benzoylation(in pyridine) tetrazonium(10)

2MH,SO@benzoylation tetrazonium

Pyridine, tetrazonium

White's aniline blue,orange G(42)

Gomori (18)

Par.F. sub. Par.

Par. andsmear

Par.

Par.

Smear

Susa

Acetone, 15 percentformaun or none

Acetoneornone

None

Gomori (19)

Bluetetrazolium (40)

Smear Modified Bourne(37)A acerbic acid None

a Section of clam in which presence of acid mucopolysaccharideaand alkaline pho.phataae and abeenceof lipids was demonstrated (57).M.B.E. - Methyleneblueextinctiontest. Par = Paraffinsection. Fros. - Frozensection.F. sub. —Forinolsublimate. F. caic. —Formolcalcium. P.AS. —Periodicacid Sebiff.

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760 Cancer Research

the cytoplasm and a small number of slightlylarger irregularly sized neutral red staining partides, mainly located in the nuclear hof.' A fewlarge refractile globules in the cytoplasm did notstain. Instillation of Sudan black stain beneaththe cover glass of supravital preparations clearlydemonstrated the lipid nature of the refractileglobules, which stained in a few seconds, and of themitochondria, which were visibly stained afterabout 10 minutes. Unfortunately, the Sudan blacksolution rapidly destroyed the supravital stainingproperties of the cell, and there was a latent periodwhen the cell was unstained and considerableshrinkage occurred. It was not possible, therefore,to visualize the exact fate of every neutral redparticle. A comparison of drawings of the supravitally stained and the Sudan black-stained cellrevealed that some of the neutral red vacuoleswere probably identical with sudanophilic granuleswhich became visible at about the same time asthe mitochondria, but were mostly confined tothe nuclear hof.

Phase contrast microscopy confirmed the existence of spherical particles of variable size in thecytoplasm but did not distinguish different types.

In sections stained by Altmann's, Regaud's,and Harman's methods for mitochondria, andthe Cajal silver, Sjovall, and Kolatchew osmicacid methods for Golgi bodies, the results werevariable ; cytoplasmic particles corresponding tothe Janus green and the neutral red staining material were present in parts of sections stained byall methods except the first, in which only a fewcoarse granules could be seen. In the less intenselystained preparations only the smaller granuleswere stained, while, as the density of staining increased, the larger granules stained around theperiphery and finally appeared as solid bodies.Even in the most intensely stained preparationsscanty unstained vacuoles were present in theperipheral cytoplasm. No vacuoles, however, werepresent in Sudan black preparations, indicatingthat the vacuoles were probably produced by thelargest cytoplasmic globules whose sudanophiliawas observed by direct staining of cells under themicroscope.

Since the standard Golgi stains failed to showany structures other than those which were revealed by supravital staining and by phase microscopy, it seems justifiable to consider the largerargentophilic and osmiophilic granules which

1 The term “nuclear hof― indicates the zone of cytoplasm

which is the site of the centrosome and the lipochondria orGolgi apparatus and is usually adjacent to a slight indentationof the nucleus.

resemble the sudanophilic, neutral red stainingbodies as Golgi bodies or lipochondria (38).

In smear preparations of tumor cells (but notparaffin sections) fixed in formol sublimate andstained with dilute toluidine blue, there were numerous dark blue cytoplasmic granules, identical in shape, size, and distribution with the supravitally stained mitochondria (Fig. 1).

In summary, therefore, at least three types ofsudanophilic material were demonstrated in thecytoplasm of the tumor cells. The smallest andmost numerous particles were mitochondria; a fewlarger granules, mainly in the nuclear hof, werelipochondria or Golgi bodies; and, until they canbe more clearly defined, the largest will be calledlipid globules.

Cytocheini@try.—Thetinctorial and cytochemicalproperties of the cytoplasmic and nuclear structures of the tumor cells are summarized in Table 2.

Nucleus and nucleolue.—Thenuclear chromatin,including the nucleolar membrane, was the onlymaterial in the cell which gave a positive Feulgenreaction. In acid solutions of aniline blue and orange G the nucleolus and parachromatin granuleshad a stronger affinity for orange G than any othercell component, and therefore contained histone(42). The nucleolus was poorly stained by tolui

dine blue in smear preparations, and the intensity of staining was not decreased by ribonucleasedigestion (Figs. 1 and 2). The nucleolus had thesame staining range as the cytoplasm in themethylene blue extinction test, and was similarlyaffected by previous ribonuclease digestion (Table2). The nucleolus, like the rest of the cell, staineddark red by the coupled tetrazonium reaction.Benzoylation eliminates the reaction of tyrosine,tryptophan, and histidine with this reagent but,according to Danielli (10), has little effect on thereactivity of compounds containing purine andpyrimidine, which may not react at all under theconditions of this test (37). After benzoylation thenuclear desoxyribonucleic acid (DNA) was notstained by the tetrazonium method, but the cytoplasm was still faintly positive. Tyrosine, tryptophan, and/or histidine were therefore present inthe nucleus, nucleolus, and cytoplasm. Extractionof the nucleic acids by treatment with sulfuricacid completely abolished the reactivity of allthe cell constituents (Table 2). Ribonucleic acid(RNA) may have been responsible for the residualstaining after benzoylation. The amount of RNAin the nucleolus and of DNA in the nucleus wasnot sufficient to react with the tetrazo compoundafter benzoylation. Confirmatory evidence of thepaucity of RNA in the nucleolus was providedby the failure of ribonuclease to decrease the in

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762 Cancer Research

tensity of staining with toluidine blue. Only themethylene blue extinction test provided any evidence for the presence of RNA in the nucleolus,and this may have been caused by the presence ofthis material in the cytoplasm, which almost invariably covered the nucleolus in sections of thethickness used here.

Lipid globules, lipochondria and mitochondria.—

The cytochemical reactions of the sudanophiliccytoplasmic components (Fig. 4) may be conveniently considered together. All three componentsgave a pink color in all steps of Cain's Nile bluesulfate method and therefore contained neutrallipid (8). In material fixed in formol calcium theyall stained with Sudan III and IV, which suggests,but certainly does not prove, that they containedtriglycerides (37). With or without fixation, thecytoplasmic particulates were colorless, monorefringent, and gave a negative Schultz reactionfor cholesterol. No preformed aldehyde or acetalphosphatide could be detected in unfixed smears.The performic acid Schiff test for unsaturatedlipids was negative, and after S days' exposure toair no oxidation aldehydes could be demonstrated.Phospholipid could not be demonstrated byBaker's acid hematin test.

As already noted, the large cytoplasmic globules stained most readily with Sudan black B;they did not stain by any methods other thanthose involving simple solution of fat soluble dyes(Table @)and probably consisted mainly of tnglyceride.

In paraffin sections no P.A.S.2-positive materialcould be demonstrated in the tumor cells. Insmears stained by the P.A.S. method and mountedin glycerine jelly, a few irregularly sized P.A.S.positive granules were observed in the nuclearhof. When such a preparation was counterstainedwith Sudan black, all the red stain was obliterated by the black. After extraction of lipids,slightly smaller, less intensely colored P.A.S.-positive granules could be demonstrated in the nuclear hof. There were, therefore, two P.A.S.-positive components associated with the sudanophilicgranules of the nuclear hof which, as alreadyshown, were the lipochondnia. One component,which could be removed by treatment with hotchloroform and methanol, was a lipid ; the otherwas resistant to treatment with lipid solvents.

Glycolipids, some phospholipids, and some unsaturated lipids are the only colorless lipids reported to give a positive P.A.S. reaction (43). Thepresence of phospholipids in any quantity wasexcluded by the negative acid hematin reactionand the failure to demonstrate acidic lipids by

2 P.A.S. = Periodic acid Schiff.

Cain's Nile blue sulfate method (Table 2). Unsaturated lipid could not be detected by the performic acid Schiff reaction, and the reaction foraldehydes secondary to the oxidation of unsaturated fatty acids was negative (Table 2). Somereduction in the intensity of the P.A.S. reaction wasproduced by bromination, but no more than occurred in a lipid-free control section containingacid mucopolysaccharide. The reacting lipid wasprobably glycolipid.

The presence of acid mucopolysacchanide andglycogen in the lipochondria was excluded becausethe P.A.S.-positive reaction was unimpaired afterhyaluronidase and diastase digestion and becausemetachromasia was absent. Acetylation in drypyridine completely blocked the P.A.S. reaction ofthe cell, but, since pynidine is a lipid solvent, thisdid not provide any information about the reacting groups in the lipid component. It did, however,confirm that 1,2 glycol groups were probably responsible for the reaction of the other componentwhich, by a process of exclusion, was either aglycoprotein or a neutral mucopolysacchanide.

In smears fixed in formol sublimate and stainedwith toluidine blue, the mitochondnia were clearlystained dark blue (Fig. 1). The staining reactionmust have been due to the presence of nibonucleicacid, since it was completely abolished by previous ribonuclease digestion (Fig. 2). The close association of RNA with the lipid particles wasshown by the absence of mitochondnial stainingwith toluidine blue after extraction of lipid.

In unfixed and formalin-fixed preparations, theGomoni technic for acid phosphatase revealedminute positive granules throughout the cytoplasm of the tumor cells (Fig. 3), reaching a maximum intensity after 1 and 2 hours' incubation,respectively. After this period some diffusion occurred, and the nuclear chromatin was alsostained. The reaction was more intense in theunfixed than in the fixed material, but the numher and distribution of the particles were identicaland suggested that the enzyme was present in themitochondnia. In preparations fixed in acetone,staining appeared after S or 4 hours' incubationand was more pronounced in the nucleus than inthe cytoplasm.

Cytoplasm.—When paraffin sections were used,the mitochondria were no longer recognizable intoluidine blue preparations and the cytoplasmstained diffusely blue or faint purple. The stainingreaction was removed by nibonuclease digestion,showing the presence of RNA. The presence ofRNA in the cytoplasm was further confirmed bythe alteration in the pH of extinction in themethylene blue extinction test after ribonuclease

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LOVE AND SHARPLESS—V&US-indUced Oncolysis - Cytology 763

digestion (Table 2) and by the results of the tetrazonium reaction already described.

Viaus-ncrzc'rzo TUMORCzuaLipochondria.—No perceptible changes oc

curred in the tumor cells until the 2d or 3d dayafter virus inoculation. At this time an increasein the number and size of the lipochondria (Fig. 5)could be demonstrated by all the methods descnibed except supravital staining, which did notshow any increase in the neutral red-staining partides. In some cells the enlarged lipochondnia appeared to fuse to form one or two larger aggregations.

In paraffin sections, however, the early stagesof the development of cytoplasmic aggregationswere not seen, and the first abnormality in hematoxylin and eosin sections was the appearance ofone or two large homogeneous eosinophil inclusion bodies in the nuclear hof. (The term “inclusion body―can be applied with some justification,since no such structure can be seen in similarlyprepared sections of uninfected tumor cells.) Thesebodies stained uniformly red by the P.A.S. method,even after extraction of all stainable lipid. Likethe Golgi bodies, their ability to stain with theP.A.S. technic was unaffected by diastase or hyaluronidase digestion, was reduced by bromination, and abolished by acetylation. They stainedfaintly with Sudan black B, and on high magnification sometimes appeared to consist of a numberof smaller granules (Figs. 6 and 7). The “inclusions― differed from the smaller lipochondnia ofthe uninfected tumor cell in that they were visiblein paraffin sections, where they acquired properties which could not be shown in smear preparations of infected or uninfected tumor cells. In sections they stained more intensely than the cytoplasm, but exhibited the same staining reactionswith the tetrazonium method, toluidine blue stainan4 the M.B.E. test, and were similarly altered byprevious destruction of RNA by nibonuclease ortreatment with strong acid. They contained,therefore, protein (amino acids) and RNA. Except for the presence of RNA and allowing forsome loss of lipid in the imbedding process, thecytoplasmic “inclusions―observed in paraffin sections had the same cytochemical properties asthe lipochondria of infected and uninfected cellsin other preparations.

Mitochondria.—In supravital and smear preparations of virus-infected cells the mitochondniawere unaltered in morphology and cytochemicalproperties. Since mitochondnia were not demonstrable in paraffin sections of material fixed informol sublimate, they may also have been in

volved in the fusion of lipid particles which gaverise to the “inclusions,―and in doing so impartedtheir RNA content to the final mass.

Lipid globules.—Coincident with the changesin the lipochondria, the cytoplasmic lipid globuleswere sometimes, but not always, increased in sizeand number.

Nucleu,@.—Asthe Golgi bodies increased innumber and size the nucleus of the cell was pushedto one side and even indented by the larger aggregations (Fig. 7). Some margination of the nuclear chromatin was seen in an occasional tumorcell with hypertrophied Golgi bodies, but for themost part no further evidence of degeneration wasdetected until the cell had undergone phagocytosis. This latter process was extremely rapidwhen the tumor was growing in muscle, and tumorcells were no longer present within 2 days of theappearance of the first abnormality. In the brain,however, phagocytosis was delayed, and “inclusions― persisted in most of the tumor cells for 3or 4 days, during which analysis of the mitoticprocess was possible.

Mitotic process.—Fromthe results presented inTable 3 it will be seen that there was a significantincrease in the percentage of tumor cells in mitosisafter virus infection. Analysis of the data revealsthat this was almost entirely the result of an increase of cells in metaphase. The percentage of

infected tumor cells in the other stages of mitosiswas decreased, though only the telophase resultsare significant. There was, therefore, a delay orpartial arrest in metaphase. Further evidence ofinterference with the process of cell division wasshown by a significant increase in the proportionof binucleate tumor cells and in the percentageof abnormal mitotic figures in the virus-infectedseries (Table 3). Clumping of the chromosomesin metaphase was the commonest abnormalityof mitosis, but scattering of chromosomes andclumping in prophase, anaphase, and telophasewere fairly frequent. The same types of abnormality were present in the uninfected and infectedgroups, and the difference between the two wasentirely quantitative.

DISCUSSION

The results of this study lend support to themore recent views of Baker on the nature ofGolgi bodies or lipochondria (3). The lipochondriaof the tumor cell consisted of spherical bodiescontaining glycolipid, and some comprised anouter lipid layer and an inner zone of glycoproteinor neutral mucopolysaccharide. Cytochemicaldifferentiation between glycoprotein and neutralmucopolysaccharide is impossible, but the co

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LOVE AND SH@PLsss—Virus-induced Oncolyth - Cytology 765

existent presence of protein suggested that theformer was more probable. A similar conclusionwas reached by Gersh (16), who demonstratedglycoprotein in the Golgi apparatus of the duodenal cells in the rabbit and guinea pig.

Supravital staining clearly demonstrated thatin the infected tumor cells some of the lipochondna did not color with neutral red. Jackson (23)has recently reported a similar loss of affinity forneutral red affecting the granules of degeneratingleukocytes.

The occasional presence of phospholipids inGolgi bodies has been described by Baker (3) andby Cain (9). Evidence for the existence of vitaminC and alkaline phosphatase in the Golgi bodiesof many different cells has been reviewed byBourne (7) . These substances could not be demonstrated in the tumor cells (Table 2).

Hypertrophy of the Golgi bodies of virus-infected cells has been reported in canine distemper(11), Rift Valley fever (14), vaccinia (31), louping

ill (21), myxomatosis (13), and fowlpox (32). The“inclusions―in the virus-infected tumor cellsshowed a remarkable resemblance to the Bollingerbodies of fowipox. In a detailed study of the evolution of the Bollinger body, Ludford and Findlay(32) suggest that the elementary bodies of thevirus are incorporated in a lipid mass derived fromthe Golgi bodies. Although the size of the N.F.T.virus is not known with certainty, the size ofother strains of St. Louis encephalitis virus isprobably 20-30 mj@(12). Thus, there is little possibility of visualizing elementary bodies or evenaggregations of particles of this virus with thelight microscope. The only evidence for the presence of virus in the “inclusions―was indirect:viz., that the presence of “inclusions―in the cellscoincided with the period when virus was demonstrable in maximal amounts in the tumor (30).

According to Palade and Porter (36) RNA islocated in the endoplasmic reticulum of the cell.Differential centrifugation (24) and cytochemicalstudies (41) have shown that it is also present inthe mitochondria. The present study indicatesthat RNA was present in diffuse and particulateform in the cytoplasm and was probably associated with the mitochondria. Support for the conclusion that acid phosphatase was present in thecytoplasm, and probably in the mitochondria, isprovided by the differential centrifugation studiesof Palade (35) and Berthet and de Duve (6). Thelatter found that 55 per cent of the acid phosphatase activity was associated with the mitochondria.

The nature of the large cytoplasmic lipid globules is obscure. Similar globules have been ob

served in the Ehrlich ascites tumor cell (28). Following the classical hypothesis that the Golgibodies undergo a developmental cycle in relationto the secretion of the cell (3), it is tempting tospeculate that the lipid globules represent thefinal stage in the process after the bodies havereleased their content of secretory material.

Although there are numerous reports of theinhibition of tumor growth by virus infection, asfar as we are aware there is no record of virusesacting as antimitotic agents. Bunyamwera virusaffected the intermitotic phase of the Ehrlichascites tumor cell and had no effect on the vanous stages of the mitotic process (28). N.F.T.virus infection, on the other hand, was responsible for partial arrest in metaphase and somefailure of cytoplasmic division. The cytoplasmic“inclusions―in the virus-infected tumor cells wereof sufficient solidity to indent and displace thenucleus. The delay in metaphase might have resulted, therefore, from mechanical obstructionto the separation of the metaphase chromosomes.

SUM.MARY

1 . A cytological and cytochemical study of the

RPL-12 tumor cell is presented. Three types oflipid particles were identified in the cytoplasm.The significance of the largest is unknown. Thesmallest were mitochondria and probably contamed neutral lipid, RNA, and acid phosphatase.The remaining particles appeared to be lipochondna and contained glycolipid and glycoprotein.

2. Infection of the tumor with the N.F.T.strain of St. Louis encephalitis virus induced anincrease in the number and size of the lipochondna, partial arrest of mitosis in metaphase, andincreased numbers of mitotic abnormalities andof binucleate cells.

3. The mitochondria of the tumor cell were

unaffected by virus infection, but appeared to fusewith the hypertrophied lipochondria in the process of paraffin imbedding and contributed to theformation of large cytoplasmic “inclusion bodies―seen in paraffin sections.

ADDENDUMDuran-Reynals has observed that fowl pox was

activated by painting the skin of chicks withmethylcholanthrene (F. Duran-Iteynals, Studieson the Combined Effects of Fowl Pox Virus andMethyicholanthrene in Chickens. Ann. New YorkAcad. Sc., 54:977—99,1952). The similarity of the“inclusions―in the RPL-12 tumor cells infectedwith St. Louis virus to Bollinger bodies suggestedthat activation of latent fowl pox might have occurred. To investigate this possibility, a 10 per

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766 Cancer Research

cent suspension of tumor, which had been infected3 days previously with NYU virus and was knownto contain “inclusionbodies,― was inoculated in0.2-mi. amounts onto the chonio-allantoic membranes of ten 7-day-old chick embryos. Two embryos were harvested daily, and no gross or histological evidence of fowl pox was observed.

REFERENCES1. BArn, J. R. The Structure and Chemical Composition

of the Golgi Element. Quart. J. Microscop. Sc., 85: 1—71,1944.

2. . The Histochemical Recognition of Lipine. Ibid.,87:441—70,1946.

8. . Further Remarks on the Golgi Element. Ibid.,90:298—307, 1949.

4. B@tanm, A. M. Method for Staining Sections of BoneMarrow. J. Path. & Bact., 56:188-85, 1944.

5. Bmmi@zr, S. H. On Presence, Properties and Distributionof Intercellular Ground Substance of Loose ConnectiveTissue. Anat. lice., 60:98—109, 1984.

6. Bnwriin'r, J., and Duva, C. DE. Tissue FractionationStudies. 1. The Existence of a Mitochondria-linked,Enaymically Inactive Form of Acid Phosphatase in BatLiver Tissue. Biochem.J., 50: 174—81,1951.

7. B0URNE, G. H. Mitochondria and the Golgi complex.In: G. H. Bourne (ed.), Cytology and Cell Physiology,2d ed., pp. 282—86.London: Oxford Univ. Press, 1951.

8. CAIN, A. J. The Use of Nile Blue in the Examination ofLipoids. Quart. J. Microscop.Sc., 88:888-92,1947.

9. . An Easily Controlled Method for Staining Mitochondria.Ibid.,89:229—81,1948.

10. D@izui, J. F. A Study of Technics for the CytochemicalDemonstration of Nucleic Acids and Some Componentsof Proteins. In: Nudeic Acid. Symp. Soc. Exper. Biol.,

No. 1, pp. 101—13.London : Cambridge Univ. Press, 1951.11. DEL R50 Howrno@, P. Alteraciones del sistema nervioso

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12. Ei@roaD, W. J., and Pauna.au, J. R. The Size of St. LouisEncephalitis Virus as Determined by UltrafiltrationAnalysis. J. Path. & Bact., 40:148-46, 1985.

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14. . Cytological Changes in the Liver in Rift ValleyFever, with Special Reference to the Nuclear Inclusions.Ibid., 14:207—19,1988.

15. Fiannu, E. R. The Destruction of Cytoplasmic Basophilia with Mineral Acids. Stain Technol., 28:9-12, 1958.

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Magnification of all figures X 2,000.

Fro. 1.—Mitochondria in tumor cells. Note faintly stainednucleoli. Smear stained with toluidine blue.

Fio. 2.—Tumor cell stained with toluidine blue after ribonuclease digestion. Chromatin and nucleolus stain more intensely than in Figure 1. Mitochondria are unstained. Smear.

Fio. 3.—Multiple granular deposits of lead sulfide in thecytoplasm and some diffuse staining of the nucleus of tumorcell. Smear fixed in formalin: 3 hours' incubation; acid phosphatase stain.

FIG. 4.—Numerous lipid particles of varying size in the cytoplasm of uninfected tumor cells. Frozen section. Sudan blackB stain.

PIG. 5.—Increased size of cytoplasmic lipid particles 2 daysafter inoculation of virus. The increase in number of particlescannot be seen from the photograph because of poor depth offocus at this magnification. Frozen section. Sudan black Bstain.

FIG. 6.—Partial fusion of cytoplasmic lipid granules in two

tumor cells 8 days after inoculation of virus. A macrophage(below) contains a fragment of lipid, probably from an ingestedtumor cell. Paraffin section. Sudan black B counterstained withneutral red.

Fio. 7.—Morecomplete fusion of lipid material to form alarge “inclusionbody.― Paraffin section. Sudan black B counterstained with neutral red.

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LOVE AND Su@RPL@ss—Virus-induced Oncolysis - Cytology 767

25. Lnrn, R. D. Ethylenic Reaction of Ceroid with Performic Acid and Schiff Reagent. Stain Technol., 27:37—45, 1952.

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80. . Studies on a Transplantable Chicken Lymphoma(RPL-12). II. Mechanism of Regression Following Infection with an Oncolytic Virus. Ibid. (in press).

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nique. 3d ed. New York: Paul B. Hoeber, Inc., 1950.34. McM@rus, J. F. A. Histological and Histochemical Uses

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87. PEABSE,A. G. E. Histochemistry, Theoreticaland Applied.Boston: Little, Brown & Co., 1958.

88. Rins, E. Zur Histophysiologie des Mausepankreas nachLebendbeobachtung, Vitalfarbung mid Stufenuntersuchung. Ztschr. Zellforsch. u. mikr. Aunt., 22:528-85, 1985.

89. RoanEu, M. Méthode de detection histochimique deslecithines. Compt. Rend. Soc. de biol., 96:1282-84, 1927.

40. SELIOMAN,A M., and Ru'rsnnrnuo, A. M. The Histochemical Demonstration of Succinic Dehydrogenase.Science, 113:317—20, 1951.

41. VENDRELY-RANDAVEL, C. Sur in presence d'acide ribonucleique au niveau du chondriome. Acts Anat., 7:226-88,1949.

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48. Wouw, M. Staining of Lipids by the Periodic-AcidSchiff Reaction. Proc. Soc. Exper. Biol. & Med., 76:588-85, 1950.

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1954;14:758-767. Cancer Res   Robert Love and George R. Sharpless  OncolysisLymphoma): III. Cytological Changes during Virus-induced Studies on a Transplantable Chicken Tumor (RPL-12

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