Viruses and Tumors in the Light of Electron Microscope Studies a Review*

Viruses and Tumors in the Light of Electron Microscope Studies A Review*

LEON DMOCHOWSKI

(Section of Virology and Electron Microscopy, The University of Texas M. D. Anderson Hospital and Tumor Institute, and Department of Microbiology, Baylor University College of Medicine, Texas Medical Center, Houston, Texas)

CONTENTS I. Introduction 978 II. General Ultrastructure of Cells 979 A. Normal cells 1 B. Tumor cells J 1. The cell or plasma membrane of normal and tumor cells ~. The cytoplasm of normal cells 3. The cytoplasm of tumor cells 4. The Golgi apparatus of normal and tumor cells 5. The centri01e of normal and tumor cells 6. Mitochondria of normal cells 7. Mitochondria of tumor cells 8. Other cytoplasmic constituents of normal and tumor cells 9. The nuclear membrane of normal and tumor cells 10. The nucleus of normal and tumor cells 11. The nucleolus of normal and tumor cells III. Ultrastructure of Virus-infected Cells and Viruses 984 A. Herpes simplex B. Salivary gland virus C. :Fowl pox D. Vaccinia E. Ectromelia and molluscum contagiosum F. Meningopneumonitis virus G. Influenza H. Anopheles A virus I. Poliomyelitis J. Coxsackie viruses K. Trachoma virus L. Varicella M. Adenoviruses N. Newcastle disease O. Infectious myxomatosis P. Nucleic acid changes in virus-infected cells IV. Ultrastructure of Virus-induced Tumors and Tumor Viruses 988 A. Rous sarcoma B. Chicken leukosis * Presented in part at the Symposium on Contributions of Electron Microscopy of Viruses and Cells to the Problem of Cancer, held during the Seventeenth Annual Meeting of the Electron Microscope Society of America, Ohio State University, Columbus, Ohio, September 9-1e, 1959. Preparation of this paper was aided by Research Grants No. C-8679 and C-4140 from the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service, and by Grant No. 94-B from the American Cancer Society. 977

1. Visceral lymphomatosis /. Neurolymphomatosis 3. Granuloblastosis (myeloblastosis) 4. Erythroblastosis 5. Adenoeareinolna of the kidney of chickens C. Chemically induced tumors in chickens D. Normal chicken tissues E. The Shope fibroma of rabbits F. The Shope papilloma of rabbits G. Mammary tumors of mice and the Bittner virus H. Leukemia and other neoplastic conditions of mice 1. Spontaneous and induced leukemia ~. Parotid gland tumors 3. Polyoma-induced tumors of mice ~. X-ray-induced leukemia in mice 5. Other tumors of mice and rats I. The Luck6 adenoearcinoma of the frog J. Human tumors of known, suspected, or unknown viral origin V. Future Prospects 1001 VI. References 1003

I. INTRODUCTION viral agents could be detected in the infected The discovery of viruses at the end of the cells and the observation that these agents can 19th and the beginning of the ~0th century has be differentiated from normal cell constituents led to the development of virology as a new branch have led to electron microscope studies of tumors of biological science which has now grown into in the origin of whieh viral agents were either one of the most prolific and stimulating disciplines known or suspected to play a part. Electron micros- of all biological sciences. Soon after the discovery copy has also proved helpful in assessing the of the so-called infectious viruses came the discov- progress of isolation and purifieation procedures ery of the first tumor-inducing viruses. The part of tumor-indueing viruses, and in the study- of played by viruses in the origin of tumors gained the relationship between the isolated viral particles recognition only slowly. Indeed, it came to be and tumors induced by the inoeulation of suspen- fully recognized only during the last 8 years (114). sions eontaining these partieles. Like any other The new interest in viruses as causative agents method, electron mieroseopy has its limitations. in cancer was brought about by the results of the Nevertheless, its applicability to other methods, application of a number of methods, some long whether ehemieal, physical, or biologieal, makes known in virology but with possibilities only re- it not only one of choice but of neeessity in the cently realized, and some entirely new. Among present and future studies of tlle relationship the methods which have proved to be very helpful of viruses to the origin of tumors, both in animals in the study of viruses in relation to tumors is and lnan. electron microscopy of ultrathin sections of normal A discussion of the contributions of electron and tumor cells and of various preparations made microscopy of viruses and cells to the problem of from tumorous tissues. cancer requires, as a first step, an answer to several Following the application of electron micros- questions. These are: (a) what is a virus; (b) what copy to tlle stu(ty of normal cells, the study of is cancer; (c) are viruses the cause of cancer; (d) cells in viral infections was undertaken. It has what is the relationship of viruses which produce led to the visualization of viruses in the infected infection to those known to cause cancer? tissues and has revealed the complicated structure a) Viruses are intracellular, infectious, poten- of these agents and tile behavior of various sub- tially disease-producing nueleoprotein en t it ies, with microscopic cell elements (luring the different only one type of nucleic acid, which reproduce stages of infection. Almost simultaneously with from their own genetic material, are unable to the application of ele(.tron microscopy to the grow and divide, and are devoid of enzymes (0~94). cytology of normal and virus-infected cells, elec- It will be seen later that eleetr()n inicroscopy tron microscopic studies of malignant cells were has confirmed, at least in part, this definition of undertaken. The comparative ease with which a vir/i~.

b) There is no sharp distinction between a past and present reviews are of importance in nornlal and a cancer cell. A gradual, progressive this rapidly developing field (216, 250, 251, 329, change from organized to unrestricted behavior 330, 366, 367, 400, 401,420, 424, 443, 474). of cells takes place in cancer, leading to the The development of suitable fixation procedures invasion of surrounding, then gradually more dis- (335, 372, 380, 417, 419), the introduction of taut cells, and to their final destruction (410). plastic as embedding material (70, 71, 326), and Thus, a distinction between a benign and malig- the development of ultrathin sectioning technics nant tumor is at best an arbitrary one. through the introduction of glass knives (282) c) Cancer, no matter of what type or origin, and of special microtomes (239, ~41, 354, 368, is known to be the result of many diverse factors, 418) have made possible a systematic study of such as genetic, hormonal, and environmental. the ultrastructure of cells. In addition, the technic Viruses are known to be one of the factors involved for the separation of cell components by differen- in the origin of tumors. The present-day question tial centrifugation (242, 344, 345), following the is in how many tumors are they one of the causa- original work of Claude (91, 92, 93), has provided tive factors. From the results of electron micro- important information about the chemical con- scope studies, so far obtained, it may be stated stitution and enzymatic activities of some of the that electron microscopy will play an important isolated cell constituents studied in the electron part in answering this question. microscope. A number of cellular components thus d) The essence of viral infection is not the acquired a new" functional and biochemical mean- disease, but the introduction into the host of a ing. These components vary in their appearance foreign entity which is able to multiply in the during different stages of the life of cells and cells of the host and reproduce other foreign in pathological conditions. Thus, gradually, cell entities, which may but do not necessarily have morphology, cell physiology, and biochemistry to produce a particular disease (294). Electron are becoming one integrated science. microscopic, biochemical, and immunological stud- Electron microscopy has contributed greatly ies have so far failed to reveal any essential dif- to our knowledge of the ultrastructure of cell ferences between infectious and tumor-inducing components and has led to a better understanding viruse< They all share a similar requirement for of their functional significance, although it has hving cells. Indeed, the tumor-inducing viruses revealed comparatively few new structural com- produce many symptoms characteristic of an in- ponents of the cell. A great deal more study is fection, and there are recent indications that some required, which should combine electron micros- infectious viruses may participate effectively in copy of the various cell components separated the development of tumors (148). by means of differential centrifugation with a Having thus answered these preliminary ques- biochemical analysis of these constituents derived tions, it is now possible to turn to electron micro- from cells in various stages of activity, in order scope studies of viruses and tumors. A proper to obtain some new basic concepts of normal and assessment of the contribution of these studies tumor cells. The structural components of cells to the part played by viruses in the origin of are not artifacts, because there exists an agreement tumors requires a thorough understanding of the between the structure of cells as seen under the ultrastructure of normal cells and viruses as seen phase-contrast and in the electron microscope. in the electron microscope. This will allow for A brief survey will be presented of the appearance a comparison between normal and cancer cells of the various components of normal cells as seen and between infectious and tumor-inducing viruses in the electron microscope, in order to make the on the morphological level. In turn this will be ultrastructure of viruses and their relationship helpful in the discussion of the part played by to these components more comprehensible. viruses in the development of tumors and in an attempt to indicate the trends and to emphasize B. TUMOR CELLS the aspects which require further clarification of No features of the submicroscopic structure this problem. of various cell constituents have as yet been found with the help of the electron microscope which H. GENERAl, I TLTRASTRUCTURE could be attributed exclusively to tumor cells. OF CELLS Any differences observed between the ultrastruc- A. NORMAL CELLS ture of normal and malignant cells are only of a There are a number of reviews on the contribu- quantitative character (106, 297). They may be tions of electron microscopy to our knowledge expressed either in the loss of submicroscopic of the submicroscopic appearance of cells. Both cell components (40, 331) or in their occasional

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. 980 Cancer Research Vol. ~0, August 1960 hypertrophy (49, lP2). It is impossible to discuss The profiles of the endoplasmic retieulum dis- viruses and their relationship to tumors as seen play tubular and vesicular structures (343). They in the electron microscope without a brief descrip- have been described as lamellar (10~), fibrillar tion of the various cell constituents in tumor (333), or eanalieular (335) structures, or as pairs cells, which will follow the description of the same of membranes (45~1). An excellent review on this constituents in normal cells. component of the cytoplasm is now available 1. The cell or plasma membrane of normal and (elS). It may vary in volume, distribution, and tumor cells.--The electron microscope has revealed structural details from one type of eell to another, the structure and dimensions of the cell membrane but is found in the cytoplasm of most cells (339). and some of its components (4~0, 4~1, 477). In It is involved in the proeess of cell differentiation some cells, such as the cells of the convoluted (340). It may be represented by circular or oval tubules of the kidney (380), the cell membranes profiles in the eytoplasm, bound by smooth mem- form deep invaginations creating extensive com- branes, and forming interconnected vesicles and partments for mitoehondria, probably for the pur- tubules (340, 341) or larger spaces described as pose of extending the function of the membranes eisternae (343). This structure may appear both to the interior of the cells (4~0). Thus, the electron as smooth (agranular) and rough (granular) mem- microscope has revealed the cell membranes in branes, aceording to the absence or presence of certain types of cells to be more extensive and dense small particles (338, 341, 343). Both types complex than it was known from the light micro- of membranes may be present in eertain types scope studies. The invaginations of the cell mem- of cells, their content being eontinuous, and the branes may also form cytoplasmic vesicles (~50, relative amount of the two types of membranes 339, 460). It is clear that a great deal more study may vary in different types of eells (340). The is required to obtain a functional interpretation small dense partieles attached to the rough-sur- of these findings, but a beginning has already faced elements represent a distinct cytoplasmic been made in the studies of fat absorption in the component (338). The granular component of cells of the small intestine (1346). The basement ergastoplasm is composed of particles, 80-300 A membrane has been particularly well described in diameter, attached to the outer surface of the in the cells of some organs (380, 1381, 4~1), and membranes and vesicles and also scattered singly found to have a characteristic structure (135~, 388). or in groups in the cytoplasm (338). Cytoplasmic It would be of interest to study the behavior basophilia is at least in part due to these granules of this membrane as well as of other cell com- (~49, 338). The cytoplasmic RNA appears to ponents in the kidney of mice during the various be confined to the granules and is not eonnected stages of infection with tumor viruses such as with the walls of the sacs or membranes (344, polyoma virus. 345). In some cells the system of membranes No differences have as yet been observed be- fills the whole ground substanee of the cytoplasm; tween the eell membranes of normal and malignant in other cells (5~, 17s 347, 348) it is concentrated cells, except for the cytoplasmic protrusions whieh in certain regions of the eytoplasm, and in still may vary in size and number in tumor cells others it may be absent or seldom encountered (1398). The new methods of staining of thin sections (889, 4~0). There are differences of opinion as for electron mieroseopy (457, 458) may further to the terminology of this eomponent, variously elucidate the structure of the cell membranes of described as endoplasmie retieulum (1363), ergas- both normal and malignant cells. toplasm (460), eytomembranes (419), or lanlel- ~. The cytoplasm of normal ceUs.--Certain re- lae (10~). Currently, the membrane eavities and gions of the cytoplasm have been found in the spaees of the eytoplasm are being regarded as light microscope to stain with basophilic dyes and a single intereonneeted system, the rough and the have, therefore, been described as the basophilie smooth membranes, simple vesicles, and the nu- component of the cytoplasm or as ergastoplasm clear envelope representing loeaI differentiation by Gamier (196). Ultraviolet absorption methods (13139, 1341, 1345~2, 456). The elements of endoplasmie have revealed ribonueleic acid in this component reticulum also eommunieate with the exterior of of the cytoplasm (88). It was first observed in cells (164, 340). The eytoplasmie membranes have the electron microscope in cells grown in tissue reeently been visualized in eertain types of mam- culture (1370) and found preferentially located malian eells with phase eontrast mieroseopy (176). in the cytoplasm (6, 5~, 6o~, 108, ~66, ~99, 13713, This makes possible an investigation of these 40~2). This component of the cytoplasm has been eytoplasmie membranes in living eells. named endoplasmie retieulum (13613, 1364, 1371) be- The ergastoplasm is the souree of the so-called cause of its appearance as a lace-like retieulum. microsomes obtained by differential ultraeentrifu-

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. DMocHowsKI--Eleclron Microscopy of Viruses and Tumors 981 gation of cells (344, :345). The microsomes are subject of much controversy among microscopists. particles of 100-150 A diameter and appear to be In the clectron microscope studies it has been nucleoprotein particles responsible for the RNA found to be a constant, highly organized region content of the cytoplasm. Combined morphologi- of the cytoplasm and independent of the endo- cal and biochemical studies of this cell fraction plasmic reticulum (101, 1013, 104, 377, 1380, 42~). in virus-infected cells may help in our understand- ttowever, according to some investigators this ing of the mode of action of viruses. component is only a continuation of the endoplas- 3. The cytoplasm of tumor cells.--The ergasto- mic reticulum (339, 340). It has been described plasm or endoplasmic recitulum has been found in a variety of cells of vertebrates (105, 107). to show great variability in tumor cells (16~, It may be present either in the centrosphere region 164, 178, 333). Strange formations of membranes (343) or scattered throughout the cytoplasm (348). have frequently been encountered in the cytoplasm The Golgi zone is composed of a system of closely of cells in tumors of viral origin, such as mouse packed, smooth melnbrancs and elongated, flat- leukemia, both spontaneous and induced, and tened, small vesicles with no granules of the Palade in all forms of the chicken leukosis complex. The type. These membranes surround a series of vacu- RNA particles are present in the cytoplasm dif- oles of varying size and shape. The vacuoles, which fusely scattered and in clusters (~249, ~50, 385). usually appear empty, occasionally contain dense, The increased basophilia of tumor cells parallels granular material. They may vary in occurrence, an increase in RNA granules (38). The swelling, number, and size (105, 218, 274, 848). It is of vacuolization, and disruption of membranes of interest that the Golgi zone has a uniform struc- the ergastoplasm (57, 115, 116, 398) have also ture in cells of the most diverse organs of different been found in inflanunatory cells (361) or in species. There is no doubt that it plays an impor- cells subjected to starvation (195). In plasmocy- tant part in the secretion of cells. The Golgi toma, the endoplasmic reticulum is well developed apparatus is involved in the formation of aerosome (359), compared with normal cells of the plasmo- (8s specific granules of the anterior hypophysis eyte type, but is dilated and filled with a clear (218), concentration and segregation of lipide ma- homogeneous substance, as in the bone marrow terial (461), and the removal of water from secre- cells in myeloma (58, 74, 75). The morphological tory or absorbed substances (105). The Golgi variations in the ergastoplasm indicate variations apparatus in vertebrates appears to be similar in the chemical properties of the so-called micro- to that of invertebrates (~73). somal fraction of tumor cells compared with thai. As in the ease of other cytoplasmic components, of normal cells. It is, however, often difficult the Golgi zone of tumor cells shows no characteris- to distingqlish between cause and effect, and stud- tic difference from that of normal cells (106, ies, like those on the influence of dyes on the 218, ~1, 250). Occasionally it has been found endoplas,nic reticulum in the early stages of car- to be enlarged in spontaneous mammary tmnors cinogenesis (369), are as yet only too infrequenl. of mice (49), hepatoma of the rat (s erythro- Such studies combined with histochemical and blastosis of chickens (37), uterine carcinoma of the biochemical methods may in due course reveal rat (38). However, in other tumors, e.g., Rous al least some of the mechanisms of carcinogenesis. sarcoma (57, 162), Ehrlieh and Yoshida aseites A study of Rous tumor cells has revealed a tumors (466), either it is not well developed or system of cisternae and vesicles (Golgi zone) in it may be absent (359). two widely separated areas of the individual cells 5. The centriole of normal and tumor cells.--Seen (16~2). It is an interesting observation, since this as a dark spot in the light microscope, it appears structure has always been found in normal cells in the electron microscope as a short cylinder in the region of the centrosome only. In cells composed of fine tubules, single or multiple (10, of this tumor, the cell membranes have been 233, ~51, 4136, 446) near the center of the Golgi found to have an unbroken connection with the zone, and resembles the structure of cilia of mam- outer nuclear membrane through the membranes malian cells (177). of endoplasmic reticulum, both smooth and rough In spite of occasional hypertrophy of the Golgi (16~). Thus, a direct communication in these cells apparatus in cells of certain types of tumors, the exists between the outside of the cells and the eentriole is the same as in normal cells (~233). nucleus (164). 6. Mitoehondria of normal cells.--Mitochondria 4. The Golgi apparatus of normal and tumor were discovered by Altmann in 1899, although cells.--Ever since the original description of a the actual name was proposed by Benda (340). structure in the r of nerve cells by Golgi, They form distinct structural units in the cyto- this component of the cytoplasm has been the plasm and can be separated in a comparatively

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. DMocHowsKI~Electron Microscopy of Viruses and Tumors 983 even in different cells of the same tumor (13, 38, described by numerous sources (5, ~0, ~9, 87, 56, ~49, ~284, 466). Changes in the structure of 109, 193, 348, 36~, 376, 380, 455, 456). These mitochondria are frequently found in tumor cells pores appear to be formed by fusion of outer and (56, 115, 116, 385, 403, 464). These changes are inner membranes, a single layer extending across by no means a rule, even in the cells of the same the diameter of the ringlike structures (164). Ma- tumor, although they have frequently been en- terial composed of ~00-A granules both within countered in the cells of tumors of viral origin. the nucleus and the cytoplasm has been shown The so-called "microbodies," dense, spherical for- as possible evidence of the porous nature of the mations, have been found in tumor cells and in nuclear membrane (12). The reality of the pores many normal as well as diseased cells (195, 380, has, however, been questioned (~1, 194, ~3~, s 384). Dense, granular masses filling mitochondria 38~). have been observed in hepatomas induced by There is no doubt that the structure of the dimethylaminoazobenzene in rats (38). There ap- nuclear membrane shows considerable variation pears to be little doubt that certain viruses develop in different types of cells. The "pores" have been within mitochondria of tumor cells, as will be found in mammalian, amphibian, insect, and pro- discussed later. Morphological studies combined tozoan cells. Numerous cytoplasmic particles are with biochemical studies of isolated mitochondria associated with the outer layer of the membrane, may perhaps be helpful in shedding at least some which appears to be a continuation of the rough- light on the mode of action of some tumor viruses. surfaced endoplasmie reticulum (339, 456). In 8. Other cytoplasmic constituents of normal and some cells the inner nuclear membrane is thicker tumor cells.--Various types of osmiophilic particles than the outer (348). have been described in cells of different types In recent high-resolution studies, the nuclear (~50), as well as fibrillar structures and secretory membrane of mammalian cells, fixed in OsO4, granules (4~0). has again been shown to be composed of two Some of the changes observed in tumor cells parallel membranes and to form circular pores are similar to those in normal cells following of 1000 A by the junction of inner and outer neutral red administration (463). Appearance of membranes (459). Low-density parts of the nucleo- gray amorphous material, osmiophilic inclusions plasm are associated with these pores and extrude of varying number, size, and complexity, and their into the cytoplasm. Thus, the distinction between vacuolization all take place in the cells injected the cytoplasmic and nuclear portions of cells does with neutral red and in tumor cells. not become as clear and obvious as previousl3~ A new cytoplasmic component, "pentalaminar considered. Nuclear membranes fixed in KMnOa body," has been described in some animal and differ somewhat from those fixed in OsO4. In human cells (306). Similar membranous structures place of the pores or annuli, circular areas of low have been observed in cells of a variety of virus- density are seen, which appear not to be true induced tumors to be described later. They have holes (194) but may be covered by material thin- also been described in Yoshida rat sarcoma cells ner than the rest of the membranes. (475). The nuclear membrane of the tumor cells is Certain previously unknown structures, de- similar to that of normal cells (~19). Bone marrow scribed as "membranae fenestratae" (398) or "an- cells in plasmoeytoma show a perinuclear space nulate lamellae" (444), have been observed in with numerous vacuoles which are connected with certain normal and tumors cells (444, 466). Their vaeuotes of the endoplasmic reticulum (358, 359). part in the cell life is as yet unknown. Various 10. The nucleus of normal and tumor cells.--The types of inclusions are present in tumor cells. They nucleoplasm does not appear as a highly organized may be an expression of secretory functions or they component when studied in the electron micro- may be degenerative in origin, comprising fat, scope. The granular material encountered meas- complex myelin figures (360). Occasionally bundles ures from 150 to ~50 A in length (1~, 17~, 364, of thin fibers, probably an expression of cell de- 380, 4~1). It is possible that the round particles generation, may be found in the cells of certain or short rods arc cross-sections of loosely coiled, tumors (63, 385, 466, 475). fine threads of chromosomes in the interphase 9. The nuclear membrane of normal and tumor nucleus (173, ~51). The thickness of chromosome cells.--The nuclear membrane has been described filaments in different stages of prophase and in as a double-layered structure (87, ~3~, 4~3). The metaphase varies from 50 A to 70 A in prophase existence of "pores" (400-500 A) or ringlike struc- to 100 A in metaphase (383). The thinnest chromo- tures in the outer sheath of the nuclear membrane, some filaments (~8 A) may be single deoxyribo- with the inner sheath being continuous, has been nuclear protein molecules (383).

The frequently observed invaginations of the in due course to the identification of the relation- nuclear membrane are not characteristic of tumor ship of structure and biochemical function, and, cells (398, 466). The chromatin, indistinguishable thus, reveal any differences between normal and from that of normal cells, has granular or fila- malignant cells. With the knowledge of submicro- mentous structures 80-150 A in diameter. No dif- scopic structure available, although far from com- ferences between chromosomes of normal and tu- plete, a study of cells infected with different mor cells have been observed. viruses has been made possible. 11. The nucleolus of normal and tumor cells.- The main constituent of the nucleolus is a system III. ULTRASTRUCTURE OF VIRUS- of coiled or lamellar structures embedded in a INFECTED CELLS AND VIRUSES homogeneous material. The structures are com- There are a number of reviews on this subject posed of small dense granules 100-150 A in di- (400, 401,468, 474). The development of a shadow- ameter (4~, 54, 7~, 364). Morphologically, these ing technic has considerably helped in the descrip- granules resemble those described in basophilic tion of the shape and size of viruses (470, 471). cells as ribonucleoprotein particles (1r 47, 337, The literature on electron microscopy of shadowed 365, 4~1). This conclusion is supported by studies preparations of viruses has been extensively re- of cell fractions which have defined, small cyto- viewed (118, 401,468, 474). Following the isolation plasmic particles rich in ribonucleic acid (355). and purification of tobacco mosaic virus by Stan- Cytochemical studies (73, 88) support the evidence ley (431), the first shadowed virus particles to obtained by electron microscopy that RNA is be photographed in the electron microscope were in fact formed in the nucleolus, diffuses through those of plants and bacteria. Under carefully con- the nuclear membrane, and contributes to the trolled conditions the method can be used for the RNA of the cytoplasm. identification of plant, insect, bacterial, and animal The nucleoli in tumor cells are frequently en- viruses, when based on the correlation of the larged, although both the matrix and the fila- number of the characteristic particles widl the mentous component are unchanged (41). No fun- results of the test for infectivity of the preparation damental difference has been found between the containing these particles. nucleolus of normal and of cancer cells. In certain By means of quantitative electron microscopy types of tumor cells the filamentous network may of shadowed preparations (18, 19, 469) a number be predominant (41, 43, 466), whereas in other of animal viruses, such as Coxsackie virus (77, types of tumor cells the matrix may be pre- 30~), foot-and-mouth-disease virus (16), and polio- dominant (41, 464). Examples of the appearance myelitis virus (17), have been identified as in- of nuclei and nucleoli in the various lesions of the fectious morphological entities. Analytical mi- squamous-cell carcinoma of the eye in cattle (141) croscopy has been employed to assess the progress are shown in Figures 1, ~t. It is, as yet, unknown of purification procedures of poliomyelitis virus whether the differences in the appearance of the (399). An absolute virus titration by means of nucleolus of certain types of tumor cells are con- electron microscopy has been achieved in the case nected with the metabolism of these cells. In of T~ bacteriophage, in which every particle was the electron microscope the cells of a number of found to be an infective unit (~9~). In the case types of tumors have been observed to preserve of influenza virus it has been possible by use of the function of the normal cells from which they electron microscopy to observe structural differ- originated (51, 76, ~84, ~98), showing products ences between the infectious and noninfectious of their activity and hypertrophy of the constitu- particles and to separate both the infectious and ents responsible for the secretion. noninfectious particles from normal host material Only the application of biochemical and electron by their absorption on the surface of red cells microscope methods to the fractional ultracen- (465). trifugation technics (413, 414) may help in the Electron microscopy of shadowed preparations development of our knowledge of the functional of viruses following different physical and chemical significance of the various cellular constituents treatment has occasionally revealed outlines of of both normal and tumor cells. The purity of an internal structure in viruses. In animal viruses, each fraction can now be checked with compara- such as vaccinia and molluscum contagiosum, rive ease in the electron microscope and the preser- the treatment with proteolytic enzymes has led vation of the various components assessed, follow- to the removal of the greater part of the virus ing physical fractionation and biochemical extrac- particle, leaving a small dense core and a thin tion. Perhaps a combination of histochemical membrane around the core. Further treatment methods and electron microscopy (~6) may lead with deoxyribonuclease led to a decrease in the

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. DMocHowsKI--Electron Microscopy of Viruses and Tumors 985 core and liberation of soluble nucleic acid (468). viruses (311, 31~, 4~5-~9) within the infected It became known that, in some viruses, bacterio- cells. Comprehensive reviews of early studies of phage, tobacco mosaic, turnip yellow mosaic, and animal viruses by electron microscopy of sections the pox viruses, the nu('leic acid is localized inside of cells infected with viruses are available (~23, a protein envelope (467). Electron microscopy 467). It is amazing to reflect on the rapid progress of shadowed preparations of viral particles gave, of these studies. It mirrors the constantly increas- therefore, a good indication of the complexity of ing interest in this type of approach to the study structure and chemical composition of viruses. of viruses and their behavior ill the infected hosts. The (Icvclopmcnt of the ultrathin sectioning The increasing use of tissue culture for the study technic has considerably increased the value of the of viruses within cells has provided a better op- appli(:ation of electron microscopy to the study portunity to follow the intracellular development of viruses. It has proved of great value in the of viruses at different times following infection. siu(ly of the structure and chemical properties This method allows for correlation of the appear- of viruses, of their appearance and behavior in ance of infected cells with the appearance of cells, as well as of their mode of development infective particles released from these cells. This and their relationshi I) to various cell constituents. is a line of approach along which greater effort IIowever, a virus particle cannot be identified should and undoubtedly will be made in an at- as such on the basis of morphology alone, but a tempt at identification of the different viruses. significant correlation must bc obtained between the nmnbcr of the characteristic particles and the A. HERPES SIMPLEX infectivity titcr, which also reveals the number IIerpes simplex virus was one of the first animal of particles per infectious dose (468). Observation viruses to bc examined in sections of the chorio- of virus particles within cells themselves does allantoic membrane (818). Characteristic spherical not allow, however, for quantitative microscopy particles of two different sizes have been found combined with bioassay. It is true that particles in the nuclei of cells. In the cytoplasm, the par- with an internal structure characteristic of virus ticles are larger (1,100-1,300 A), have a dense particles differ from any known normal cell com- core, but arc surrounded by a double membrane. ponent. The similarity in size and structure of These observations suggest a certain growth mech- particles observed in the infected tissues to those anism, as yet not fully demonstrated (313). Par- of known virus particles do not necessarily indicate ticlcs of 300-400 A present in the nucleus arc that they are the causative agent of the particular considered the first stage of infection. Recently, disease. This applies to both infectious and tumor- the development of a strain of herpes simplex inducing viruses. Ilcnce, the term "virus-like" virus in IIeLa cells and in human amnion cells particle encountered with increased frequency in has been described (319, 3o~1). F(mnation of ag- literature on electron microscopy of sectioned tis- gregates of granules (~00 A), crystals of particles sues. The identification of the characteristic par- with a single membrane, and particles with double ticles as virus particles in sections of tissues has membranes (1,~00-1,300 A), all take place ill the been helped by the results of the following ap- nucleus. Similar granules have been observed in proaches: tissue eulture study of virus-infected the nuclei of cells infected with adcnoviruses (67) cells; differential chemical staining procedures of and with influenza (3~0). The particles appear such cells combined with their study by electron to enter the cytoplasm either by disruption of the microscopy; separation of the eharaeteristie par- nucleus or reduplication of the nuclear membranes. tieles from these cells and the correlation of their A profusion of lamcllac with porelikc structures number with the infectivity titer; correlation of has also been observed in the cytoplasm of cells their morphologie appearance with infectivity fol- infected with herpes simplex (3~1) and attributed lowing various chemical treatments to nonspecific degeneration (3~23). A conclusion Electron microscopy has recently led to the was reached that the development of herpes sim- visualization of tobacco mosaic virus in the in- plex virus may bc completed in the nucleus. It fected leaves (300) and to the elucidation of the would have been of interest, in this elegant mor- structure of tobacco mosaic virus, which is com- phological study, to correlate the nmrphological posed of a central rod of RNA and a surrounding observations with the presence of the virus by protein envelope (468). It has been helpful in means of bioassays. the morphological assessment of chemical pro- In an attempt to correlate the appearance of eedurcs of degradation and reassembly of tobacco the presumably infected cells as seen in the elec- mosaic virus (467). It. has also revealed the ap- tron microscope with the amount of virus released pearance of bacterial viruses (~69, o~70) and insect at the time of sampling, monolayers of monkey

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. 986 Cancer Research Vol. ~0, August. 1960 kidney cells inoculated with a known herpes B D. ~rACCINIA virus-cell mixture were studied (~279). Ten hours Morphologically similar results have been ob- following infection, characteristic particles with tained in sections of the chorioallantoic membrane single (600-1,000 A) or double membranes (1,900- infected with vaceinia virus (15~, 314, '315). The 1,800 A) were observed both in the nucleus and particles (1,600-3,000 A), like those of fowl pox cytoplasm, as well as outside the cytoplasm of virus, have a "nueleoid" body, similar in structure cells (437). This may indicate simultaneous forma- to that seen after enzymatic treatment of the tion of particles in both locations. At the time of virus particles. The suggested mode of develop- release of the infective virus the charac'teristic ment of these viruses would be as follows: The particles were detected on the external surfaces finely granular regions form small units, the so- of cells. The difference between this observation called "nucleoids." The nueleoids increase in size, (379) and those previously described (313, 3~1) and at first a single, then a double, limiting mem- may be due to different host cells, different strains brane appears. A characteristic complex structure of the virus, and the inherent errors in sampling has been shown in the vaccinia particles (167, for electron microscopy. 356). Electron microscopy of fluorocarbon-purified B. SALIVARY (~LAND VIRUS preparations of vaccinia virus, combined with bioassays, has provided additional evidence that Characteristic particles present in cells infected these particles are, indeed, vaccinia virus (o~47). with both human and mouse strain salivary gland There is a need for more cytochemical and electron virus appear to be morphologically similar and microscope studies of vaccinia virus. Such studies are also similar to those in the cells infected with may help to elucidate biochemical changes taking herpes simplex and herpes B virus. Particles, pre- place in virus-infected cells. sumably the salivary gland viruses, have been observed t)oth in the nucleus and in the cytoplasm E. ECTROMELIA AND ~[OLLUSCUM of the infected cells (~293). The cytoplasmic par- CONTAGIOSUM ticles (1,600-1,800 A) have been found associated Intracelhllar changes in cells infected with eel ro- with the Golgi zone, ergastoplasm, and mito- melia (301) and mollusc'urn contagiosum (~200) chondria (~93). Thus, presumably, the virus dur- have been found similar to those taking place ing its development may be connected with all in the cells infected with vaccinia (199). Lamellar three components. structures and characteristic whorl-like formation s C. Fowl Pox have recently bccn described in the eytoi)lasm In sections of the chorioallantoic membrane of Ehrlich aseites tumor cells infected with ectromelia. Recent studies on sections of cells infected infccte(l with fowl pox virus, two kinds of particles with molluscum contagiosum have revealed details were obscrve(l (314). One type was composed of structure of these particles anti of other struc- of an eccentrically located, (tense core, surrounde(l tures interpreted as developmental forms of the by granular material and by a single membrane, virus (143). These studies have shown a striking and t)rescnt in the cytoplasm of cells. The other morphoh)gical similarity between the particles in type, with a larger central ('ore surrounded by molluscum contagiosum and those observed in granular material and a double membrane, was fibroma of rabbits and in vaccinia. present near the periphery of the cyt()plasm and in the cxtraccllular spaces. The suggestion was F. ~{ENI1N'GOPNEUMONITIS VIRUS put forward that the core develops from the con- ]n the cytoplasm of cells infected with men ingo- densation of granular material, which increases pneumonitis virus, round and oval structures have in size and loses some granularity and that a been observed containing a central dense body membrane is formed around the core. It will and varying in size from "2,500 to 3,000 A (197). be seen that a similar course of events appears Larger particles (3,000-4,000 A), with two or three to take place in some of the tumor-inducing limiting membranes around a dense core, have viruses. These stu(lics again, like those previously also been found in cytoplasmic vacuoles, "l,s well mentioned, indicate the necessity of (.ombining as particles with a single membrane and two or a purely morphological approach with bioassays, three (tense centers. All these structures may be in order to establish with certainty the appearance (tcvclopmental forms of the virus, but further :and identity of the infective particles and their studies are required to establish the actual tootle mode of (leve]opment. of development of this virus.

G. INFLUENZA (~65). In a most recent study of human amnion Electron microscope studies of cells infected cells infected with the virus, swelling and break- with viruses of the influenza group at first proved down of mitochondria, swelling of ergastoplasm, to be inconclusive (~7). Later studies of the and disappearance of mitochondria have been chorioallantoic membrane infected with influenza noted (441). In addition, virus particles (~40 A) virus have revealed on the surface of entodermal have been found in crystalline order in the cyto- cells rods (500-600 A) and spheres (600-700 A) plasm (441). Such studies require correlation of within 6 to 44 hours after infection (3~r No the morphological appearence of the infected cells changes have been observed in the nuclei or cyto- with the virus production before final identification plasm of the infected cells. It still remains to of these particles can be made, but there is reason- be ascertained whether, as suggested (3~r the able hope that they do represent poliomyelitis spherical particles are infective and the rods are virus. noninfective units. In a study of sections of pellets J. COXSACKIE ~TIRUSES from purified influenza preparations combined Intracytoplasmic crystals composed of ~50-A with infectivity and hemagglutination tests, the particles have recently been observed ill cells active virus has been found to consist of spherical infected with Coxsackie viruses (1317). Thus, an- particles (700 A) with an internal structure shown other case of intracytoplasmic crystals formed by previously (3~), and the incomplete and heated mammalian viruses is available. virus to consist of spherical particles without the dense center (66). These findings agree with the K. TRACtlOMA VIRUS results of studies of metal-shadowed preparations The essential similarity of the structure of the of influenza virus particles and with the data trachoma virus, in what may be developmental on the chemical nature of the virus (~53). Studies forms different from those of other viruses, has along these lines should be ador~tcd for other recently been reported (307). viruses and tumor viruses. L. VARICELLA H. ANOPHELES A VIRUS An electron microscope study of skin biopsy Inconclusive results have been obtained from specimens and of human embryo cells grown in a study of sections of Ehrlich aseites cells infected tissue culture and infected with variceUa virus with Anopheles A virus (189, 190). Particles (550- has revealed in the nucleus of these cells particles 650 A) consisting of a hollow core (~50-800 A), (700-1,100 A) with an internal dense core and surrounded by a complex dense shell (1~0-170 A), single membrane and in the cytoplasm particles have been observed in the infected cells. These (1,000-1,600 A) with two membranes (451). It particles have been found intimately associated appears, therefore, that the possible mode of de- with endoplasmie retieulum and in the vicinity velopment of the virus resembles to a certain of lipide bodies in the cytoplasm. In view of their extent that of herpes simplex and herpes B viruses. presence in the uninfected Ehrlieh aseites cells (188), it appears certain they are not the virus. ~I. ADENOVIRUSES They may be a by-product of viral activity or Studies on HeLa cells infected with adeno- the result of nonspeeifie cellular damage. Similar viruses have revealed nuclear inclusions composed structures have been found in other mouse tumors of spherical particles (650 A), packed in close and will be discussed later. hexagonal arrays (~8-~30, ~71, 318, 45~). It should be mentioned that crystalline formations I. POLIOMYELITIS were found in cells infected with other viruses The first investigations of monkey kidney cells (81, 1378). In spite of the relatively small size, infected with poliomyelitis virus (889) revealed the intranuclear particles show an internal dense small particles in the nuclei of the infected cells, zone and a limiting membrane. The size of these with the disappearance of nucleoli and damage particles (640-700 A) as seen in sections is smaller to the cytoplasm. In a study correlating the mor- than that obtained by ultrafiltration (800-1,~00 phologic appearance of monkey kidney cells in- A), ultra-centrifugation (1,000 A), or electron mi- fected with poliomyelitis virus with the time of croscopy of metal-shadowed purified preparations virus release, no virus particles could be found-- (e40). only a large number of osmiophilic bodies (1,~00- It is of interest to note that, while the charac- 1,500 A) present in the cytoplasm of the infected teristic particles could only be found in up to cells during the time of the most rapid virus release ~5 per cent of the infected cells, in the light micro-

scope practically all cells exhibited tile cytopathic (11.3) and of interference of the cell with viral effect Q230). This effect may primarily be a toxic maturation l))'ocess (4). phenon~enon (100), since the eyt()pathie effect of influenza virus on IteLa cells is not accompanied O. INFECTIOt:S MYXOX~ATOSlS by the reproduction of the infectious form of the It is known that the causative agent belongs virus (~238) and viral multiplication and eyto- to tile pox group of viruses. In the cytoplasm pathogenicity can be dissociated in the case of of cells of myxoma, particles varying in size from IIeLa cells and poliomyelitis virus (1). 800-8,500 A have been found. The largest par- The development of changes in HeLa cells tieles nmy be the mature infectious agent, while infected with adenoviruses and the appearance the smaller are probably noninfective units (170). of the characteristic particles have been correlated There is no doubt that additional studies of this with the growth curve of the virus (~75). Thus, virus are required. there is no doubt that these particles are adenovirus. The nature of dense eytoplasmie bodies P. NUCLEIC ACID CIIANGES IN and membranous structures observed in the in- VIRUS-INFECTED CELLS fected cells remains unknown. Similar structures Another approach which should be taken up have occasionally been found in normal cells (380), in correlation with electron microscope and im- normal fibroblasts of human embryo lung cultures munological studies of viruses, by means of the (~75), in the cells of Shope fibroma (43), and in fluorescent antibody technic (99), is fluorescence the cells of a number of other tumors of viral microscopy of the nucleic acid changes in cells origin. infected with viruses and tumor viruses (14, 15, A study correlating histochcmical and electron 448). The use of the acridine orange fluorescence microscope findings has shown that the intra- technic has shown qualitative and quantitative nuclear crystalline aggregates present in the cells changes in the distribution of nucleic acids within infected with adenoviruscs and composed of or- the nucleus and the cytoplasm of virus-infected dered arrays of viral particles are Feulgen-positive cells (3~8). Thus, DNA has been found in the cyto- and, therefore, contain DNA (67). The application plasm of cells infected with pox viruses; an altered of Fculgen-azure staining method allows for dif- RNA pattern in the c rtoplasm of cells infected ferentiation between viral and nuclear DNA (67). with West Nile encephalitis virus; the presence In this manner, in addition to the Feulgen-positive of RNA in the nucleus, distinct from that of the crystals, also Feulgen-negative crystals counter- nucleolus, in the case of influenza virus; and the stained with azure have been observed in the occurrence of DNA aggregates in the nucleus in nuclei of infected cells. They may represent a adenovirus infections (14, 3~8). By use of proteo- counterpart of the noninfeetive protein of plant lyric enzymes and DNase, the viral DNA could viruses which lacks nucleic acid (816). Similar be differentiated from the nuclear DNA (3~8). studies should be applied to other viruses and Thus, a useful indication of the nucleic acid struc- tumor viruses. They certainly go some way toward ture of tumor viruses present in tumor cells and characterization of the changes in the infected purified preparations could be obtained. A step cells, although they do not identify the particles in this direction has ah'eady been made and will as the causative agents of the particular disease. be discussed in the part on Rous sarcoma virus. N. NEWCASTLE DISEASE IV. ITLTRASTRI:CTUIIE OF VIRITS- Characteristic inclusion bodies have 1)een (le- INDUCED TUMORS AND scribed in the cytoplasm of EhrIich ascites tumor TITMOR VIRUSES cells infected with Newcastle disease virus (4). There are a number of reviews of the early Elliptical inclusion bodies of large size, composed attempts at morphological identification of tmnor- of multiple laminae and (lense granules, have inducing viruses (113, 400, 401, 468). The early been observed in the cytoplasm within 6 hours attempts at identification of the mammary tumor- following infection. Vacuolization of ('ells, changes inducing virus (Bittner virus) in extracts of mam- in mitochondria, and breakdown of cells oceur mary tumors, and in milk of virus-harboring mice, after 10 hours following infection. IIowever, no yiehted contradictory results as to the size of characteristic virus particles could be observed, particles seen in metal-shadowed preparations and the conclusion was reached that virus particles (~03, 350, 351). Following the application of the may be the granular material within the cytoplas- spray-drop technic (19), no difference could be mic inclusions (4). The inclusion bodies may be seen between the particle populations from ex- an expression of defense mechanism of the cell tracts and milk of virus-carrying and those from

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. DMocHowsKI~Electron Microscopy of Viruses and Tumors 989 similar extracts of apparently virus-free strain thinness of the sections, so that the particles mice (e48). Nevertheless, a correlation was found can be missed because of the plane of section cut- between the presence of particles of 300-A diam- ting, but this does not appear to be the full eter in the extracts of tumors and the infectivity explanation. It should also be remembered that of such extracts (138). In spite of this, doubt not all cells examined need be malignant, although remained whether the Bittner virus was in actual they are similar in morphological appearance. It fact visualized in these studies. The application is possible that the virus undergoes a develop- of the thin sectioning technic, as will be seen mental cycle in the cells and is present in a later, has considerably helped in the progress detectable form for only a short time. There is of the identification of this agent and gives hope no doubt that the number of particles in the cells of a final satisfactory result. of a Rous tumor and the nunlber of cells with Electron microscopy of metal-shadowed prepa- particles are influenced by the age of the tumor rations of characteristic particles from plasma and probably by the age of the host. Tumors of chickens with the so-called erythromyeloblas- a few days old have been found to contain more tosis (myeloblastosis) has led to the identification cells with particles than tumors 3 or 4 weeks old of the agent of this neoplastic disease (153). A in chickens of the same line injected with the same good correlation has been obtained between the standard Rous virus preparation (Dmochowski, number of particles and the reciprocal of the latent Grey, and Bryan, unpublished). This point re- period of tumor induction. The same method has quires further investigation, and tissue culture led to the identification of the rabbit papilloma methods would appear to be most promising for virus (79, 408, 409). Nevertheless, the internal this purpose. morphology of these viruses remained to be ob- A statistically significant correlation found be- served by the ultrathin sectioning technic of the tween the tumor-inducing activity of the virus infected cells and of high-speed centrifugal pellets obtained from the cells of a Rous tumor and the obtained from infected material. percentage of cells containing the characteristic particles was a first step toward the identification A. Rocs SAUCOMA of these particles with the Rous virus (160, 161). Numerous particles of uniform size and density Approximately 50 virus particles were found to were observed in the cytoplasm of Rous sarcoma initiate a tumor under the experimental conditions. cells grown in vitro and photographed in the elec- A further step in this direction was made by the tron microscope as whole cells (94). This original observation of the characteristic particles in the observation was eventually confirmed (45), but sections of high-speed centrifugal pellets from very the structure of the virus remained unknown active tumor extracts (~). Further, a close cor- until the introduction of the sectioning technic. relation has been established between the number Gaylord (198) was first to observe the presence of particles present in the sectioned pellets and of particles of a characteristic structure, similar the tumor-inducing activity of the same material to that of herpes simplex virus in the intracyto- (165, 166, ~0). The identification of the particles plasmic vacuoles and outside the cells. He also in Rous sarcoma tumors was especially necessary described pores in the nuclear membrane of Rous in view of the observation of similar particles in tumor cells and the scarcity of mitochondria. certain cells of normal chickens (34, 385). By This observation was confirmed and extended the application of the fluorocarbon precipitation (57). Particles (700-800 A) with an internal dense method (~01) to extracts of Rous tumors, followed core (300-400 A) and two concentric membranes by differential high-speed centrifugation, pellets (384) have been found in two-thirds of the ex- containing the characteristic particles (700-750 A) amined tumors (57). An additional membrane have been obtained, and their infectivity has been surrounding the electron dense core has been correlated with the presence of the particles (166). demonstrated in these particles by Epstein (163). These pellets served as a source of material for It is of interest that the particles have not been the cytochemical study of the Rous virus. found in all the examined tumors or in all cells An electron microscope study of high-speed of the tumors in which they have been observed. centrifugal pellets from fluorocarbon-treated Rous The number of cells with particles varied from tumor extracts, combined with fluorescence mi- 1 in 50 to 1 in 3,000 cells (161, 16~). The question croscopy of these pellets stained with acridine of why all tumor cells do not contain the charac- orange, before and after treatment with RNase teristic particles is one constantly encountered and DNase, has revealed that Rous virus particles in the electron microscope studies of all tumors contain ribonucleic acid (168, 169). This study of viral origin. In part it may be explained by the has also shown that ribonucleic acid is localized

in tire (lense central core or "nucleoid" of the shouh.l be mentioned that in most cases exposure particle. It appears of interest that the part of of chickens to infection has resulted in the develop- the pellets, known from electron microscopy to ment not only of visceral lymptmmatosis but also contain amorphous material, has been found to of erythroblastosis. It is not known whether these contain DNA, while a similar part of the pellets two neoplastic conditions are produced by two from fluorocarbon-treated, uninfected chick cho- different or one multipotent virus. In electron rioallantoic membranes failed to show DNA (169). microscopic studies of shadowed preparations from Although biological tests on Rous virus particles plasma of fowls with lymphomatosis, particles treated with RNasc have not as yet been carried have been observed similar to those obtained out, this study indicates the manner in which from plasma of chickens with erythromycloblas- studies on other tumor viruses should bc carried tosis (406). Electron microscopy of sections of out. Thus gradually, morphological observations, spleen from chickens with natural or experimental- combined with cytochcmical methods, are leading ly induced visceral lymphomatosis has revealed to the knowledge of the chemical composition in the cytoplasm of cells of these organs spherical of tumor viruses. or spheroid particles varying in diameter from Electron microscopy of cells grown in tissue 640 to 8~20 A (116, 118, 119, 1~25). The particles culture and infected with llous virus may prove have an internal dense core (300 A), surrounded helpful in the elucidation of its mode of develop- by a less (lense zone and by an outer membrane mcnt. The original observation that Rous virus occasionally seen as double membrane. They are can induce neoplastic change in vitro (14~) has present in the cytoplasmic vacuoles, in inclusion been confirmed in chick fibroblasts grown in tissue bodies in the cytoplasm, an(t in the intercellular culture (~296, 387) anti should be the subject spaces (Fig. 3). The inclusion bodies may be of of an electroil microscopic stutty. Tissue culture mitochondrial origin, since they frequently contain studies of Rous sarcoma virus should be made parts of internal, mitoehondrial membranes and caster by the development of a precise and quanti- are surrounded by a membrane sometimes seen tative assay of the virus (447, 45,3, 454). as double membrane. The particles have been Particles identical in size and appearance with observed in apt)roximately one out of 50 cells those of ]{()us sarcoma virus have also been found examined. In addition, changes in the cytoplasm in vacuoles and on the cell surface of Fujinami of cells have been observed, such as vacuolization, mvxosarcoma (~298). Particles of similar structure breakdown of mitoehon(tria, of endoplasmie re- but of a larger size (l,100 A) have been observed tieulum, and of cellular memt)ranes which, al- in the cells of Murray-Begg endothelioma (,385). though not sl)eeifie, have frequently been found The nature of these not very numerous particles in association with the presence of inclusions and has not as yet been established. The relationship characteristic viral structures (10~5). A study of of Ilous virus particles to those observed in various sections of the spleen of young, control chickens forms of the chicken lcukosis coinplex presents of t!-.-" same breed (so-called line 15 of the U.S. an interesting anti imt)ortant problem. Regi(mal Poultry Research Laboratory) has failed to reveal similar particles (lO~5). B. CIIICKEN LEUKOSIS ~2. Xeurolymphomalosis.--Particles similar to The neoplastic conditions that are now classified those in visceral lymphomatosis have also been as the chicken leukosis complex were the first tu- observed in the cells of spleen and bone marrow mors discovered to bc induced by filtrable agents. of chickens with neurolymphonmtosis (`38), but Some tumors of the fowl leukosis complc• such not in the affected nerves of chickens with neu- as visceral lymt)homatosis (8,3, 84), osteopetrosis rolymphomatosis (Burmester, Dmochowski, and (83), erythroblastosis, and myeloblastosis (30) have Grey, unpublished). been transmitted by cell-free preparations. Elec- :3. Granuloblastoai.~ (myeloblas[osis).--Electron tron microscope studies of metal-shadowed par- ntieroseopic studies of shadowed preparations from tMes from plasma of diseased chickens led to the plasma of chickens with myeloblastosis combined identification of the agents responsible for eryth- with t)ioassays demonstrated a correlation between roblastosis, myeloblastosis, and lymphomatosis the number of the characteristic particles (1,~00 A) (30), but they failed to reveal their internal strut:- and the infectivity titer of the preparations (405). ture. By this method, however, no internal structure 1. Visceral lymphomato,~is.--The natural occur- could be observed in the particles. The difference rence of visceral lymphomatosis is widespread, between the size of the shadowed particles and that the disease being transmitted via the egg and 1)y of particles seen in the sections of organs of chick- contact with the infected environment (84). It ens with myeloblastosis appears to be due to

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. DMOcHOWSKI--Electron Jlicroscopy of Viruses and Tumors 991 differences in the technics, each of which produces swollen mitochondria and osmiophilic bodies vary- obviously different amounts of artifacts. ing in size and shape ("gray bodies"), some in Extensive electron microscope studies (118, the process of vacuolization and surrounded by 1s 1~6) of sections of organs of chickens a membrane (169). In many of these bodies, with with granuloblastosis have shown in the cells outlines of what could be interpreted as remnants of the affected organs vacuolization of the cyto- of mitochondrial cristae, virus particles have been plasm and alterations in the mitochondria, in the found. In the cytoplasm of myeloblasts from e- endoplasmic reticulum, and in the cellular mem- to 4-week-old cultures, fully developed particles branes (Figs. 4, 5). Dense osmiophilic bodies, have been found in smaller number than in the surrounded by a single or double membrane, have cells from young cultures. In the circulating myelo- been foun

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. 99~ Cancer Re~.earch Vol. o~0, August 1960 and chemical tests. There appears to be no doubt have been observed between the agents or erythro- that the characteristic particles are the virus of blastosis and granuloblastosis (30). myeloblastosis (granuloblastosis). These morphological studies indicate the useful- 4. Erythroblastosis.--Avian erythroblastosis may ness and at the same time the limitations of a be caused by the "R" strain of En gelbreth Itolm (30) purely descriptive approach. They have shown and by "RPL-I~" strain, which originally induced the particulate nature of the agents of these dis- mostly visceral lymphomatosis but, after serial eases, already known from previous studies (30), passages, produced also erythroblastosis when giv- the similarity of changes in the cells of the affected en in large doses to susceptible chickens. In low organs, and the possibility of a similar mode of doses, both strain "R" and "RPL-I~" induce development of the agents in these diseases. This, visceral lymphomatosis (86, ~I4). The virus of of course, will require further investigation for visceral lymphomatosis may be a "wild type" which electron microscopy of cells grown in vitro virus (84), because, in contrast to erythroblastosis, and infected with the agents may be particularly it has maintained itself in chickens for many useful. The morphological observations indicate years. The relationship of visceral lymphomatosis also the necessity of biochemical studies of mito- virus to the virus of erythroblastosis "RPL-I~" ehondria isolated from the cells of the organs and "R" strains, as well as to other viruses of affected by these diseases. This may lead to the the chicken leukosis complex, is one of great elucidation of some of the biochemical changes interest. In electron microscope studies of sections in the infected cells and perhaps to a better under- of organs with these two types of erythroblastosis standing of the mode of action of these viruses. (1~0--1~4), changes have been observed in the It is interesting to note that viral activity has cells of spleens of chickens (Fig. 8) infected with been found in the cytoplasmic but not in the nu- erythroblastosis strain "RPL-12" and strain "R" clear fraction of cells in chicken erythroblastosis similar to those seen in visceral lymphomatosis (171,450). Combined morphological and biochem- (1~5) and in granuloblastosis (1~6). In erythro- ical studies of the various fractions of cells in the blastosis, caused by either of the two strains, the diseases of the chicken leukosis complex appear changes in the cytoplasm are accompanied by the to be particularly interesting and may prove very presence of osmiophilie bodies, similar to "gray rewarding. bodies" (69). These bodies are frequently sur- Similar results in electron microscope studies rounded by single or double membranes (Fig. of sections of the affected organs of chickens 9) and occasionally show broken-down eristae with erythroblastosis have been reported by other of mitoehondria (1~4). These bodies of varying sources (35, ~61, 26~). Particles similar to those size and shape contain finely granular, osmiophilie described in the sections of cells have also been material in various stages of vaeuolization and found in sections of ultraeentrifugal pellets from what could be interpreted as various develop- infective plasma of chickens with erythroblastosis mental forms of the characteristic particles. These (44). Thus, evidence is available that the particles particles have also been observed scattered in seen in sections of various organs of chickens with the cytoplasm near broken-down membranes of erythroblastosis are the virus itself. the osmiophilie bodies. They have also been found Centrifugation studies of material obtained in vacuoles (Figs. 10, 11), probably at least in from chickens with diseases such as visceral lym- some instances derived from the osmiophilie bod- phomatosis, osteopetrosis, and others may be ies, among characteristic "whorl-like" formations somewhat more difficult. Extracts of infected or- in the cytoplasm (Figs. 10, 11), and in the inter- gans may have to be employed in view of the cellular spaces. The particles in erythroblastosis difference in the viral content in the plasma of strain "R" vary in size from 600 to 760 A (1~4), chickens with these diseases and of chickens with and from 650 to 830 A in erythroblastosis strain granuloblastosis. The application of fluorocarbon "RPL-I~" (1~3). The general appearance and deproteinization (~01) to tissue extracts, which internal structure of the particles have been found has already proved helpful in Rous virus purifica- similar in all four diseases, with some variation tion (166), combined with differential eentrifuga- in size of the particles observed. The particle tion and electron microscopy, may lead to the size in all four diseases has been found to overlap, isolation of the agents from the other forms of the greatest variation in size being found in par- chicken leukosis. This undoubtedly will help in tieles lying outside the cells in these four neoplastic further ascertaining the relationship of the agents conditions. A similarity in size and structure does of the different forms of chicken leukosis. not necessarily indicate identity of the agents, 5. Adenocareinoma of the kidney of chickens.-- as, indeed, chemical and serological differences Chickens given inoculations of material containing

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. DMocuowsKI--Electron Microscopy of ~ruses and Tumors 993 the virus of myeloblastosis frequently develop of the spleen and bone marrow of about 10 per adenocarcinoma of the kidney2 Cell-free extracts cent of normal chicken embryos (33, 34) and in of this tumor induce adenocareinoma of the kid- fibroblasts of normal chickens grown in vitro (179). ney, visceral lymphomatosis, osteopetrosis, and Since visceral lymphomatosis is widespread among only occasionally myeloblastosis. 1 Electron nficro- chickens and is transmitted through the egg (85), seopic study of sections of this tumor has revealed bioassays would have proved helpful in establish- particles of a structure similar to that of virus ing the nature of these particles. At the present particles in myeloblastosis (Fig. 1~), but varying time, the nature of these particles in norina[ in size from 600 to 1,400 A. They have been tissues must remain a subject of speculation. In observed in inclusion bodies in the cytoplasm of similar studies carried out on sections of spleen tumor cells and in the intracellular spaces (135). from normal young chickens of the so-called line At present it is not known whether there is a con- 15, no particles resembling virus particles could nection between the different size particles and the be found (117-119, 1~-1~6). This may be because various neoplastic conditions induced by cell-free the line of chickens are free of visceral lymphoma- extracts of this tumor. In all morphological studies tosis. of virus-induced tumors a similar study of suitable control material is an obvious requirement. Need- E. THE SHOPE FIBROMA OF RABBITS less to say, the presence of characteristic particles In 193~ Shope discovered that a fibroma of in tumor tissues and their absence in the corre- rabbits is caused by a virus (411), and in the fol- sponding normal tissues do not indicate that the lowing year he demonstrated the viral etiology particles are the causative agent of the disease. of infectious papilloma of rabbits (41r In the Further, the observation of morphologically simi- light microscopic studies ]%ulgen-positive inclu- lar particles in normal tissues does not necessarily sion bodies have been described in the paranuclear indicate that the particles in tumor tissue are not area of the cells of fibroma tumors (97). Electron the agent of the disease. A brief discussion of microscopic studies of metal-shadowed prepara- electron microscope findings in normal chicken tions of particles from fibroma have been carried tissues is, therefore, necessary. out by Lloyd and Kahler (~88). In the electron microscopic studies of sections of both benign C. CHEMICALLY INDUCED TUMORS IN CHICKENS and malignant fibroma tumors (43, ~4), the Golgi The cell-free transmission of chemically induced zone, the nucleus, and nueleoli have been found tumors in chickens is doubtful, since the original hypertrophied, and the mitoehondria showed signs claims could not be confirmed in numerous at- of breakdown. In addition, in the paranuelear area tempts under strictly controlled conditions (for of the cytoplasm of some tumor cells homogeneous. literature, see 113). An electron microscopic study zones of finely granular material have been ob- (187) of transplanted chicken tumors, which origi- served, frequentlsT containing elongated, parallel nated from a tumor induced by dibenzanthracene, lamellae about 70 A thick. In some of these areas. has shown changes in the nucleus and cytoplasm spherical particles (~,~00 A) have been found, sur- of these tumors but no convincing evidence of rounded in part or entirely by a single or double- structures resembling virus particles. Vacuoles and membrane and containing material similar to that aggregates of fibrils (170 A) have been found in of the zones. The changes in the paranuelear the nucleus and, in the cytoplasm, clusters of region correspond to the Feulgen-positive inclusion tubular structures (450 A in diameter) and nu- bodies in the light microscope. In an attempt to merous small spherical or elongated particles with study the developmental cycle of the Shope fibro- a dense double membrane (187). It is doubtful ma virus, tissue culture of virus-infected cells whether the observed changes are due to the direct was the obvious method of choice. Electron micro- action of the carcinogen, because the tumors were scope studies of sections of normal rabbit fibro- transplanted for many generations Additional blasts grown in vitro and infected with fibroma studies along these lines should be carried out virus were, therefore, carried out (97, 98, 180). in an attempt to investigate further the relation- Small, strongly Feulgen-positive areas, gradually ship of carcinogens to tumor viruses and the changing into pyronin-positive areas, were found possible activation of latent viruses by the various within 4 hours after infection. The first virus. carcinogenic factors. particles surrounded by a single menlbrane could be found in the infected cells after 8-10 hours. The- D. NORMAL CHICKEN TISSUES number of virus particles gradually acquiring dou- Particles similar to those described in neoplastic ble membranes increased considerably after ~4~ tissues of chickens have been found in sections 1Burmester, private communication.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. 994 Cancer Research Vol. '20, AugusL 1960 hours. After several days crystal-like structures The possible steps in the development of the were found in the cells. Finally, in old infected virus should be the subject of a study in vitro, cells, characteristic, concentric membranes of an along the lines carried out on fibroma virus. Only onion-like appearance could be observed. Unfor- when a system is employed in which most, if tunately, these morphological observations have not all, cells are known to be infected with the not been combined with tests of tissue culture virus can a true sequence of events be learned. material for the titer of viral activity. The changes, as observed by electron microscopy It is of interest that no difference could be during the various intervals of time following found between the appearance of particles present infection, may then be correlated with the virus in the so-called benign fibroma and malignant sar- production as shown by a quantitative and precise coma (g8) of rabbits. There is a striking morpho- assay of the virus. A study along these lines logical similarity between the particles in Shope has been carried out on myxolna virus (89) fibroma and the virus particles of the pox group (e00, 314). The morphological data, now combined G. MAMMARY TUMORS OF MICE with other previously reported data, give support- AND THE BITTNER VIRUS ing evidence that fibroma virus is a member of The early attempts (110, 113) at identification the pox group of viruses (181). This may serve of this virus by study of metal-shadowed particles as another example of the limitations of a purely in preparations of tumor extracts and of milk morphological study of viruses and cells infected have already been nlentioned. Particles of possible by them, since these viruses, although morpho- viral origin were first observed in mammary tumor logically apparently very similar, exhibit entirely cells grown in vitro (874), but could not be studied different biological behavior (143). In molluscum with any accuracy because the sectioning technic contagiosum, in addition to the changes observed had not been developed at that time. in the cytoplasm of cells infected with pox viruses, Soon after the introduction of the uttrathin dense osmiophilic aggregates have been found in sectioning technic, characteristic particles were the nucleus of cells from biopsy specimens and observed within and outside the cytoplasm of attributed to the action of the virus (144). mammary tumor cells of mice known to carry the agent, as well as in some tumors from apparently F. ThE SHOPE PAPILLOMA OF RABBITS agent-free mice (111). Since then several groups Electron microscopic studies of metal-shadowed of investigators have reported similar pavticles particles in preparations of highly purified tumor in tumors of mice with the agent and in some tu- extracts were reported some time ago (~63, 408, mors of agent-free mice, but seldom in normal 409). Recently, electron microscopic studies have mammary gland tissue of virus-carrying mice. been reported on sections of tumor tissues (both In the mammary tumors two types of particles fresh and stored in glycerine) and on at least partly were found, larger size particles (700-1,400 A) purified ultracentrifugal pellets from tumor ex- within the cells and in the lumina, and smallc,', tracts (310, 439). Characteristic particles (~50- vesicular ones (600 A average) within the cells 350 A) have been found in the tissues and the (11,, 115, 116, 137). In other studies, similar pellets. In peripheral cells of the tumor the par- vesicular particles were found in the cytoplasm ticles appear to be more pronounced in the finely of tumor cells, and the larger, more complex par- granular regions of the nucleolus, but are not pres- ticles only at the surface of the cells in microvilli ent in the nucleus or the cytoplasm of the cells and in extracellular spaces (~4, 35, 39, 4~, 49, 51). (439). In cells closer to the surface the particles The smaller, vesicular-type particles have an os- are distributed throughout the nucleus showing miophilic, double, membrane-like structure and pronounced margination of chromatin. In some electron-lucent center, and the larger particles tumor cells at the surface the nuclei are closely have a double limiting membrane, surrounding packed with the characteristic particles, while a clearer zone, which in turn surrounds a central in other cells with the nuclei absent pools of par- or eccentric (lense core (51). It has been suggested titles surrounded by thick keratinized shells are that the smaller particles become the central body present. There is a suggestion that the particles of the larger one and that the particle cytoplasm ~wiginate from the nucleolus. This in itself is of is contributed by the cytoplasm of the cell during :great interest, since relatively little attention has extrusion of the particles through microvilli (49, been paid to the nucleolus as a possible site of 51). The larger particles have been observed in ~arcinogenic action. The case of Luck6 frog renal loci of condensed cytoplasm, which could be de- carcinoma, to be described later, has been rather scribed as viroplasm and in inclusion-like bodies an exception. (111-113), which may correspond to those seen

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. DMocHowsKI--Electron Microscopy of Viruses and Tumors 995 in the light microscope (~15). The development tumor-inducing activity of extracts of such tumors of particles, therefore, may be completed before produced agreement in 70 per cent of cases (115). they leave the cytoplasm (357). It is, however, It is obvious that other technics should be utilized impossible to discuss factually the development in attempts at identification of the Bittner virus. of these particles from the study of sections of One of these is the tissue culture technic and an- tumor cells. Only tissue culture studies of suscep- other the study of purified preparations of the tible c~lls infected with the virus, undertaken at virus from mouse milk. different intervals of time following infection, may In the electron microscope, visible changes could solve the problem of their development. Similar not be seen in mammary tissue rudiments or adult particles have also been found in hyperplastic, mouse mammary epithelium grown in vitro and alveolar nodules in mammary glands of agent- treated with virus preparations from the milk carrying mice (357). In histologically indistin- of agent-carrying mice (~76-~78). The growth guishable prelactating tissue from pregnant virus- of the virus apparently without tissue changes carrying mice of the same strain, the particles was supported in mammary rudiments but not have been found only occasionally (357). These in the mouse mammary epithelium, as shown nodules have been described as "viral lesions" in bioassays. The difficulties involved in this type distinct from histologically similar prelactating of ,~tudy may be associated with the growth and tissue (357). It has not as yet been determined maintenance of cultures of mammary epithelimn, whether the nodules are induced by the virus which should be derived from young susceptible or merely provide an environment favorable to mice, in view of the known resistance of older virus multiplication. These partieles have also mice to the virus. The possible slowness of any been found in prelactating tissue of apparently morphological effect induced by the virus, in view agent-free mice of similar origin (357). The sig- of its known long latent period, may be another nificance of this finding remains to be ascertained, source of difficulty. A study of spontaneous mouse especially in view of the observations on pellets mammary tumors in short-term cultures (~76) from milk of apparently agent-free mice which has revealed a greater number of the characteristic will be described. It should be noted that mam- particles in these tmnors after 5-1~-day growth mary tumors do arise occasionally in apparently in vitro than in the original material. They have agent-free mice, and extracts of these tumors been found only outside the cells and in the inter- have in some eases shown tumor-inducing activity cellular spaces, with the smaller vesicular particles (115). Particles of similar morphological appear- present in the cytoplasm (276). No relationship ance have also been demonstrated in mouse mam- could be established between the smaller and larger mary tumors by others (~56, 442). Similar particles particles (~76). The process of formation of the have recently been reported in a methy]cholan- larger particles was found to be similar to that threne-indueed carcinoma of the uterine cervix of influenza virus (3~) and erythroblastosis virus of mice from an agent-carrying strain (4zt9). ($5). The characteristic particles present in mouse It appears from the results of bioassays that mammary tumors can easily be distinguished from the Bittner virus at least survives, if not multiplies, the casein or lipoprotein particles of milk present in cultures of embryonic mouse skin treated with in the lactating glands of mice (51, 11~), since the agent-containing milk (~76). However, viral par- latter never show an internal structure and are ticles could not be found in the cells of tissue much more variable in size. A recent extensive cultures, although material from these cultures in- study on lactating breast tissue of agent-carrying duced mammary tumors in susceptible mice (~76). mice has again failed to demonstrate viral particles The virus could not be maintained in tissue culture in that tissue (~45). of tumor cells beyond the 3d month of explantation The problem of the identification of these par- (~76). An electron microscopic study of such tumor ticles is one of the toughest yet encountered in cells, which failed in bioassays to reveal the pres- this type of study. It would be unwise to draw ence of the virus, would have been of interest. It any conclusions from the observed quantitative has recently been shown that the characteristic differences in the number of particles seen in the particles are present in the cells of mammary tu- tumors of virus-carrying and virus-free mice (~4, mors grown in vitro for over 90 transfers (445). 51, 11~), since tremendous, quantitative differ- However, the identification of these particles will enees have been found in the particle count in be possible only on the basis of a correlation of the mammary tumors of agent-carrying mice. An at- presence and number of the particles with bio- tempt at correlation of the presence or absence logical activity of extracts of the mammary tu- of the particles in agent-free tumors and of the mors grown in vitro.

In a recent valuable attempt at characterization H. LEUKEMIA AND OTHER ~EOPLASTIC of the particles observed in mamlnary tumors CONDITIONS OF ~:IICE (809), the size of the active particle, on the basis 1. Spontaneous and induced leukemia.--A new of filtration through gradocol membranes, diffu- interest in the possibility of the viral origin of sion, and deuteron irradiation of virus-containing leukemia and allied conditions was raised by the mouse milk, has been found to be in the region discovery during the past few years of viral agents: of 1,000 A. In addition, a smaller size particle responsible for the induction of leukemia, parotid (~00-S00 A) has also been found to be infective. gland tumors, malnmary tumors, and subcutane- The smaller particle may correspond with the ous sarcomas in mice by Gross (~07-r Stewart dense core and the larger with the whole particle (43~); Stewart et al. (433-435); Friend (191); (809). Whether the smaller particles are the nu- and others (~04, ~05, ~95, 303, 395, 396). The cleic acid of the virus remains to be ascertained. different types of leukemia and, even more surpris- Recent experiments have shown that nucleic acids ing, the extraordinary variety of tumors, as well from tobacco mosaic and a number of mammalian as the inflammatory and degenerative changes, viruses are able to carry the activity of the par- induced by the cell-free filtrates of neoplastic ticular virus (7, 8, 95, 96, 18~-184, s 480). tissues opened up a new, fascinating challenge Further experiments are required to answer the to the biologists, morphologists, and biochemists question whether particles of s A, present interested in neoplasia. Indeed, it brought into in lyophilized tumor tissue (851) or in milk (309), the fold of oncology many specialists of the various are indeed the nucleic acid of the mammary tumor- branches of biological science, whose new interest inducing virus. is a timely and most welcome support for cancer There is no doubt that milk is the best source research as a whole. of the Bittner virus (11~). Electron microscopic As an intial step in an attempt at characteriza- studies of high-speed centrifugal pellets from de- tion of the properties of the virus or viruses fatted and decaseinated milk of agent-carrying present in the cell-free preparations from the dif- strains of mice have shown the presence of charac- ferent types of leukemia, electron microscopic teristic particles (Figs. 1S, 14) similar to those studies of sections of organs of mice from strains previously observed in mammary tumor cells (Fig. with a high incidence of spontaneous lymphatic 16) (133, 184). These pellets have shown high leukemia have been carried out, as well as of mice tumor-inducing activity. Similar particles with with leukemia induced by cell-free preparations only a small amount of amorphous debris have of: mouse leukemic tissues (115-117, 130). Certain also been found following fluorocarbon treatment changes have been observed in the cytoplasm of the pellets (Fig. 15). Tumor-inducing activity of the leukemic cells, the nucleus as a rule re- of the fluorocarbon-treated material has been maining unaltered or showing comparatively few found similar to that of the pellets from de- changes. The changes consisted of vacuolization fatted and decaseinated milk (184). The particles of the cytoplasnl due to the formation of vacuoles may, therefore, be associated with the tumor- in the cytoplasm itself or due to alterations in inducing activity. Final identification of the par- the ergastoplasm (endoplasmic reticulum) which ticles as the Bittner virus will, however, be jus- showed disruption of its membranes; swelling, tified only if a correlation is obtained, in ex- breakdown of internal structure, and vacuoliza- periments now in progress, between the particle tion of mitochondria; filling up of mitochondria count and one titer of tumor-inducing activity. with finely granular, osmiophilic material; and Similar results have been obtained following fluo- breakdown of cytoplasmic membranes. Osmiophil- rocarbon treatment of mammary tumor tissues ic bodies of different size have been observed from agent-carrying mice and of culture media in the cytoplasm with characteristic particles with- from explants of these tumors (488). An unex- in these bodies. These particles, resembling virus pected observation of the presence of similar particles, have been found in varying numbers particles in high-speed centrifugal pellets from within the cytoplasm and in the intercellular both defatted and decaseinated and fluorocarbon- spaces, but never in the nucleus (Figs. 17-19). treated milk of apparently agent-free, genetically The particle size ranged from 570 to 1,650 A. identical mice will also require an explanation The average diameter of the particles revolved before the identification of the particles as the around the following means: 650 A, 850 A, and Bittner virus can be accepted. It is hoped that 1,100 A. The outer membrane frequently appeared bioassays of these preparations of milk from agent- as a double membrane, and the nucleoid was found free mice, which are now in progress, will help frequently surrounded by an additional membrane in the final elucidation of this complex problem. (Fig. ~0). The appearance of the particles in both

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. DMocHowsKI--Electron Microscopy of Viruses and Tumors 997 spontaneous and induced leukemia is similar. The induction of parotid gland tumors in mice with particles in induced leukemia, with an average cell-free extracts of mouse leukemic organs has diameter of 850A and 1,100A, appear to be received confirmation from a number of sources somewhat larger in size. With the exception of (145, 146, ~83, 473). In a morphological study inclusion bodies and the characteristic particles, (117, 1~7, 1~9) of the parotid gland tumors in- the changes are similar to those reported in tumor duced by cell-free preparations of leukemic organs cells grown in .vitro and described as nonspecific of mice, the cells of parotid gland tumors have (3~3). shown few signs of cellular breakdown. Vacuoles Similar particles have also been observed by in the cytoplasm, filled with homogeneous light others in approximately 50 per cent of cases of material and surrounded by dense osmiophilie mouse lymphatic leukemia, both spontaneous (50) material, have occasionally been observed, with and induced (48). In these studies the characteris- the ergastoplasm showing few changes (Fig. ~3). tic particles have been divided into three groups Structures resembling virus particles have occa- according to size and ultrastructure: Type A sionally been found, usually in the intercellular (approximately 750 A) of "doughnut" appearance, spaces and in vacuoles of the cytoplasm, but composed of two concentric membranes; Type seldom in inclusion-like bodies in the cytoplasm B, extracellular, (800-1,100 A), similar in structure itself (Figs. ~1, ~). The particles (650 A) show to the characteristic particles in mammary tumors an internal dense zone, centrally or eccentrically of mice; and Type C, mostly extracellular, (600- placed, surrounded by a pale zone which, in turn, 1,500 A), variable in structure and composed of is surrounded by a membranous structure. The a big, dense center surrounded by one or two comparative uniformity of the size of the particles membranes with an electron-translucent core (50). observed in parotid gland tumors and their limited Inclusion bodies in the cytoplasm of cells have number is of interest in view of the greater number not been described in this study. Particles similar and wider range of size of the particles found in in appearance (800-850A) have been reported spontaneous and induced leukemia in mice. Tile in lymphatic leukemia of other strains of mice observation of particles in parotid gland tumors (11) and in lymphosarcoma of mice (11). The resembling virus particles has been reported from different types of particles present in mouse leu- other sources (48). It appears that a more exten- kemic tissues may be developmental forms of the sive study of parotid gland tumors induced by same virus, or they may, indeed, represent several cell-free preparations of mouse leukemic organs different types of viral agents. The importance should be undertaken. of another approach, such as tissue culture com- The separation of the leukemia-inducing agent bined with morphological and biological studies, from the parotid gland tumor-inducing virus by is again obvious. The absence of these particles ultraeentrifugation and ultrafiltration and by oth- in lymphoid tissues of normal control mice (50, er means (~09, ~10) has already been mentioned. 180) cannot in itself denote the nature of these Electron microscopy of sections of pellets obtained particles. It is clear that a quantitative study by differential eentrifugation of cell-free extracts of these particles is urgently required. Similar of mouse leukemic organs has shown particles particles (Type "A") have been described in other similar in size to those found in parotid gland types of mouse leukemia (~34, ~35, ~37) and in tumors (117, 1~7, 1~9). The pellets, when resus- plasmocytoma (s These morphological studies pended, induced an incidence of 11 per cent of are an important, but only an introductory step parotid gland tumors in mice (1~9). Additional in the study of the leukemia-inducing virus or information along these lines is required to estab- viruses in mice. Morphological observations on the lish a correlation between the number of particles localization of the possible leukemia virus particles observed in the pellets prepared from similar main the cytoplasm of leukemic cells have been terial and the titer of tumor-inducing activity. strengthened by the finding that the cytoplasmic It should be pointed out that no changes have fraction of these cells contains leukemia-inducing been observed in the nucleus of parotid gland activity (~80). Although this does not establish tumors induced by cell-free preparations of mouse the identity of these particles, it suggests another leukemic organs in any of the studies so far re- avenue of approach toward their identification. ported. It is an important point which will be ~. Parotid gland tumors.--The separation of the discussed again in connection with the studies leukemia-inducing agent from the parotid tumor- on parotid gland tumors induced by polyoma inducing virus in mice has first been reported preparations. by Gross (209, s and also by others (80, 434). 3. Polyoma-induced tumors of mice.--Stewart The original observation of Gross (~09) on the and her associates induced a great variety of

tumors in mice (434), hamsters (159, 433), and Electron microscopic studies of metal-shadowed rats (158) by means of extracts of leukemic organs preparations from tissue culture fluids of mouse or of parotid tumors of mice, following their embryo cells (CSItBi/f), on which extracts of passage on monkey kidney, or mouse embryo a parotid gland tumor and of a leukemic lymph tissue grown in vitro. It remains to be seen whether node, both obtained from Dr. Gross, have been the agent of Stewart, described as polyoma (156), t)assagcd, showed particles of 400-450 A, and of is the same as that of Gross but increased in 5~0-6'20 A when in solution (~64). A correlation potency following serial passages in tissue culture. has hecn noted (~66) between the number of these The p()lyoma agent has also been shown to induce particles and the hemagglutination titer of the cytopathic changes in mouse embryo cultures tissue culture fluid. It is, therefore, possible that (157). either two or more viruses are involved in polyoma A study of sections of nlouse embryo cells or that one of them may hc a passenger virus with cytopathie changes, following passage unconnected with the disease. The results of elec- of I)olyoma virus, has revealed the presence of tron microscopy of polyonm-infcctcd cells point characteristic particles measuring s A, ehiefly to possible differences in the findings in cells in the nucleus (Figs. I~4, ~7), but occasionally derived from mice of different strains, as shown in the eytoplasm and outside the cells (~, 46, in the presence of only one type of particle in 131, 1,3~). The whole nucleus of some cells may mice of some strains (NIH and Af), and of two he filled with these particles (Fig. 28). Fluids types in mice of other strains (sublines of C3H from such cultures hemagglutinated guinea pig strain). These observations require further investi- red cells and also induced parotid glan(l and gation. manmmry tumors and hemorrhagic disease in It is of interest that electron microscopic studies NIH mice. (.'ontrol cultures which failed to reveal of parotid gland tumors, induced in CSII strain these particles in their cells have failed to show mice by cell-free preparations of leukelnic tissues, hemagglutinating activity and tumor-inducing ac- have failed to reveal characteristic ~70 A particles tivity. Similar particles have been reported in in the nucleus and showe(t only 650 A particles the nucleus of other (C3II) monse embryo cells both within and outside the cytoplasm of the (46) and in the cells of a nmrine lymphoma strain cells. At this moment, it is a matter of speculation infected with polyoma (~2~2). In addition, in both whether parotid gland tumors of similar histologi- types of cells the occasiolml presence of larger cal structure may bc induced by two different partMes (500-600 A) in the cytoplasm has been t.vpcs of virus. Mammary tumors induced by reported. Similar particles have recently been ob- t)olyoma differ histologically from tmnors induced served in polyoma-infected embryo cells from C3II by the Bittner virus. So far, in these tumors in mice of several sublines (CgtIf/Gr, C3ItZC,/Bi) NIlI-Swiss mice only the small, intranuclear par- (1)moehowski and Grey, unpublished). It appears ticles have been seen (13~). The studies on poly- that, in the embryo cells of certain strains of oma-infccted tissues, and on tumors induced by mice infected with polyoma, only the snmller this virus, are a good cxaml)le of the usefulness size of intranuclear particle is encountered, while of electron microscopy, but lhey also indicate in the embryo ('ells of other strains of mice par- the limitations of such an approach. The necessity tMes of a larger size are also observed in the of correlating the presence of the different types eytoplasnl. This is an interesting observation which of particles in the various tissue culture substratcs should be further explored. is obvious. There is no (loubt that a l)laquc assay In mammary an(l parotid gland tumors induced for the polyoma virus (147, 390, 391, 435) will in Nlll-Swiss mice with the polyoma-infeeted be most helpful in establishing the correlation mouse embryo culture fluids, the characteristic of tumor-inducing activity with either one or particles (s A) have been found in the nucleus more characteristic particles. Electron microscopic and occasionally in the cytoplasm of cells (13s studies of mouse embryo cells grown in vitro Similar particles have also been observed (135) and treated with extracts of leukemic organs from in the nuclei of kMney cells from mice infected nlice with spontaneous leukemia, when carried with polyoma (Fig. ~6). out at various time intervals following the treat- It wou](l be premature to conclude, on the ment, may perhaps be helpful in shedding some 1)asis of the available evidence, that the ~70 A light on the possible origin of these partieles. particles are the polyoma virus. Additional experi- 4. X-ray-induced leukemia in mice.--In view ments are needed to establish a correlation be- of the recent discoveries of leukemia-inducing tween the infectivity titer an(t the number of these viruses, the problem of the relationship of radia- particles present in the tissue culture preparations. tion energy to these agent s is one of great interest.

Initial examination of the submicroscopic struc- arrays and tubular structures have occasionally ture of leukemic organs of mice with x-ray-induced been observed in the cytoplasm of cells from leukemia (116, 117) has been confirmed and ex- Ehrlich ascites tumors (403, 404, 466, 476). A tended to the submicroscopic structure of leu- study of the various strains of Ehrlich ascites kemic organs of mice with leukemia induced by tumor cells has revealed particles (550-700 A), cell-free preparations of leukemic organs of mice similar to those described as developmental forms with x-ray-induced leukemia (1s The changes, of the Bittner virus (51), in association with the in both types of leukemia, are similar to those endoplasmic reticulum (3) in ~5 per cent of the already described in spontaneous leukemia (le8). examined cells. Similar structures have also been A difference, probably quantitative, has been found in the cytoplasm of cells of the melanoma noted between the appearance of cells in spontane- tumor $91 (106), Friend's leukemia (~34), a trans- ous leukemia and that of cells in x-ray-induced plantable granulocytic leukemia of mice (116), leukemia and in leukemia induced by cell-free and in a transplantable mouse plasma-cell tumor filtrates from x-ray-induced leukemia. The differ- (~5~). Somewhat larger particles have been ob- ence is based on changes (shrinkage, karyolysis, served in both the cytoplasm and in the nucleus fragmentation) in the nuclei of cells in the two of cells of other Ehrlich ascites tumors (475). latter types of leukemia (le8) These few examples may serve as an illustration Although no structures resembling virus par- of the restraint with which any findings related ticles could be found in the cells of x-ray-induced to the morphology of tumor cells should be inter- leukenlia (Fig. ~5), they have been observed in preted. Particles showing double membranes with the cells of leukemia induced in the same strain no internal core need not immediately be inter- of mice with cell-free material from leukemic preted as virus particles, since morphologically organs of mice with x-ray-induced leukemia (P28). they do not represent what has now become ac- These particles (400-650 A) are present in inclu- cepted as infectious virus particles. sion-like bodies (Fig. ~9) and imvacuoles in the cytoplasm of cells (Fig. 30) as well as in the inter- I. THE LUCK]~ ADENOCARCINOMAOF THE FROG cellular spaces. Cell-free preparations from x-ray- Luck6 (~90) was the first to describe an adeno- induced leukemia have been found to induce leu- carcinoma of the kidney in the leopard frog (Rana kemia in suitable, susceptible, nonirradiated mice pipiens) and attributed its causation to a virus (1~8, ~1~, s Serial passages of these prepara- (r Transmission of this tumor by cell-free tions lead to an enhancement of the activity of the filtrates has successfully been carried out (149, virus, as shown in a higher tumor incidence at 151). In an electron microscopic study of the an earlier age (el3, ~86). In the light of these tumor (174), cells of one-third of the examined biological experiments and of the electron micro- tumors have been found to show characteristic scopic studies of organs of mice with leukemia changes. In the nucleus of tumor cells small os- induced by cell-free filtrates of x-ray-induced leu- miophilic bodies, some with tails, have been ob- kemia, it appears that radiation has activated served outside or inside spheres surrounded by a latent virus. The possibility of such a relation- a single (immature particles) or double membrane. ship has previously been suggested (386). How- In the cytoplasm of cells particles (900-1,000 A) ever, a quantitative correlation between the char- have been found with a central dense core (850- acteristic particles and the leukemia-inducing ac- 400 A) surrounded by a double membrane. The tivity is still required to identify these particles extracellular particles contained yet another mem- as the causative agent of this type of leukemia. brane surrounding the double-walled particles. In 5. Other tumors of mice and rats.--Although addition, the cytoplasm contained bundles of os- no particles resembling virus particles could be miophilic filaments (~50 A in thickness and 4-5 observed in experimentally induced mammary tu- Iz in length), which may be a reaction of the cell mors in rats (398), such particles (700 A) have to the presence of the virus. been found in a transplantable mammary tumor The developmental cycle of the Luck6 virus of the rat (246). However, their viral nature could will be established, if electron microscopy is carried not be established in the transmission experi- out along the lines of light microscopic studies ments (s Characteristic particles (1,400-1,700 of cultures of amphibian kidney epithelium ex- A) have been described in the cytoplasm of cells posed to malignant cells of the frog carcinoma from spontaneous hepatomas in mice of some (150). These studies have extended previous find- Bittner virus-carrying strains (178), but their re- ings (149) on the appearance of morphological lationship to the Bittner virus is doubtful (110). alterations in the nucleolus, revealed by the pres- Characteristic particles (580A) in hexagonal ence of Feulgen-positive material during the initial

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. 1000 Cancer Research Vol. ~0, August 1960 stage of infection. Exogenous DNA granules have it is often very difficult to find virus particles in been found attached to the nucleoli, and their mammary and parotid gland tumors of mice in- number was correlated with the malignancy of duced by polyoma. Application of electron micros- the original tumors. At a later stage, the DNA copy to human breast tumors maintained in vitro granules have been found in an increased volume by means already described (~79) may perhaps in the cytoplasm (150). Similar cytochemical in- offer a better way of ascertaining the presence vestigation of tissue culture material, treated with of virus particles in these tumors. the virus, should be combined with electron mi- Following the introduction of ultrathin section- croscopy and biological studies of the material ing technic, the morphological elements of the at different time intervals following infection. This circulating human blood have been extensively approach may prove helpful in the characteriza- investigated (59, ~89). In circulating blood cells tion of the virus and may provide information structures have been observed similar to those on the interaction between the virus and various seen in other types of cells (53, 55, ~06, 353). cell constituents. No significant differences could be observed between the ultrastructure of plasma cells and that J. HUMAN TUMORS OF KNOWN, SUSPECTED, of cells in multiple myeloma (~66), or between OR UNKNO~VN VIRAL ORIGIN myeloblasts, lymphoblasts, or granuloblasts and As yet there appear to be no electron micro- corresponding normal cells (60-64, ~67). The pres- scopic studies of sections of common warts. Char- ence of characteristic inclusion bodies in a certain acteristic particles were observed in metal-shad- number of leukocytes in three cases of human owed preparations from common warts (305), and myeloid leukemia was noted in one study (61). there is a strong indication that these particles Another study along similar lines has, however, may be the causative agent. failed to demonstrate a difference between the Inclusions but no structures resembling virus circulating blood elements from normal and mye- particles have been described in the cytoplasm loid patients (55). In spite of excellent means of cells of two cases of human laryngeal papilloma of comparison available from earlier (~06) and (304). Cytoplasmic inclusions of possible viral more recent studies (s no structures resembling origin have also been reported in a case of myxo- virus particles could be visualized in the circulat- sarcoma (~84). An extensive study of sections ing blood cells from normal or leukemic patients from 91 cases of human breast carcinoma has failed or animals. It should be remembered, however, to reveal particles resembling virus particles in that in leukemia of chickens (erythro- and myelo- these tissues (~17). Lipide inclusions and aggrega- blastosis) known to be due to a virus, it is extreme- tion of chromatin have been noted in the cells ly difficult to find virus particles in the circulating of human breast tumors comparable to similar blood elements, although the virus is present in formations in molluscum contagiosum (143, 144), large amounts in the plasma of leukemic chickens. renal adenocarcinoma of the frog (174), and in In a study of biopsy material from lymph varicella infections (451). Such condensations of nodes and bone marrow of patients with different chromatin have also been found in the cells of types of leukemia, no characteristic changes could normal skin (~17). In the cytoplasm of breast be found in the malignant cells (76). Following tumor cells osmiophilic inclusions of varying size a preliminary examination of sections of biopsy and structure, probably originating from mito- material from the lymph node of a patient with chondria, have also been found. Hyperplasia of acute lymphatic leukemia (116), a more extensive the Golgi apparatus, with vacuoles containing study of biopsy material from lymph nodes of particles (400 A), probably of secretory nature, patients with acute lymphatic, acute and chronic has frequently been seen (~17). Similar particles monocytic, and acute myeloid leukemia, lympho- have also been found in the epithelial cells of a sarcoma, and Hodgkin's disease has been carried normal uterus (3~7). All the observed changes out (117, 136, 140). This study has revealed in the ultrastructure of breast tumor cells could be changes in the cytoplasm of cells of all biopsy described as an expression of quantitative but specimens (Fig. 31) similar to those already de- not qualitative differences between the tumor and scribed in mouse and chicken leukemia. These normal breast cells. The studies of human breast changes have also been found in the cells of lymph tumor cells by the electron microscope have failed nodes from five patients with non-neoplastic dis- to yield results similar to those of mouse mammary eases. In addition, particles of a characteristic tumors. These observations need not necessarily structure, similar to that of virus particles, have contradict the possibility of the viral origin of been observed in the cytoplasm (Fig. 33) and in- human breast cancer. It will be remembered that clusion bodies (Fig. 3~) in the cells from lymph

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. DMocHowsKI---Electron Microscopy of Viruses and Tumors 1001 nodes of three out of six eases of acute lymphocytic studies of the specimens grown in vitro, may be leukemia examined, in one case of acute myeloid of great help in this respect. but not in one case of chronic myeloid leukemia, Electron microscopic examination of sections and in one out of two cases of lymphosarcoma. of cells from acute and chronic leukemia (~57-~60) In all cases, the particles are of similar size (900 A) has revealed characteristic particles (700 A) in and appearance, have a central dense core, an the cytoplasm of the cells from acute monoeytic outer pale membrane, and are surrounded by leukemia, and also vesicular particles (450 A) in two membranes. These particles may represent tile cells from acute myeloid leukemia. It is inter- a virus unconnected with human leukemia. esting to note that these structures have been An increased granularity and vacuolization of observed in the cells from circulating blood, but the cytoplasm and formation of cytoplasmic in- their nature requires additional study. clusions leading to the occasional breakdown of In both benign and malignant rectal polyps, cells have been observed by means of phase mi- Feulgen-positive cytoplasmic inclusions have been croscopy in fifteen out of ~4 cases of leukemia described (~85); but no virus particles have yet and lymphoma from which lymph node material been observed in these inclusions. This type of was grown in tissue culture. Similar study of tumor requires further investigation, in spite of biopsy material from the lymph nodes of four the difficulties in finding virus particles even in nonleukemie patients has failed to reveal any tumors of known viral origin. changes in the cells of lymph nodes grown in It is gratifying to see the gradual, although vitro. Electron microscopy of cells front a ease slow, development of interest in the study of the of malignant lylnphoma grown in vitro, which submicroscopic structure of various types of hu- showed the described changes, has revealed in man tumors. This could not have taken place inclusion bodies in the cytoplasm characteristic without a background of knowledge of the ultra- particles (Fig. 34) similar to those seen in sections structure of normal and tumor cells, especially of the same and of other biopsy specimens (1S6). those from tumors of known viral origin, and with- The results of bioassays in mice of cell-free extracts out the knowledge of ultrastructure of known of lymph node specimens from leukemic patients, viruses. It is doubtful, at the present stage of of tissue culture material from the (.ells of these our investigations, whether electron microscopy specimens showing the characteristic changes, and will be used as a diagnostic tool for human cancer, from the cells of nonleukemie patients have proved but it will certainly contribute to the knowledge to be disappointingly equivocal. The inei(tence of the ultrastrueture of human tumors and help of leukemia and other tumors has been found to in chemical and tissue culture studies of these be approximately similar in animals given inocu- tumors. lations of leukemic and control material (136). V. F UTUIIE PROSPECTS There appears to be little doubt about the presence of an agent in the biopsy specimens from some Electron microscopy has contributed greatly cases of leukemia and malignant lymphoma, as to the knowledge of the submicroscopic constitu- shown by electron microscopy of both the original ents of both normal and malignant cells. By biopsy specimens and of those grown in tissue correlation of the morphological and chemical ap- culture, as well as by the behavior of some of the proach much information has been obtained on specimens when grown in vitro. The mice of the certain cellular constituents, particularly on the strains used appear to have a latent, probably endoplasmie reticulum (344, 315, 414-416), which polyoma-like agent. This, of course, does not ex- is abundant in cells in which large-scale synthesis clude the use of mice of other strains, especially of proteins takes place. From such cells, a par- those free of polyoma virus. Future bioassays ticulate fraction described as mierosomes has been may show that there is no suitable laboratory obtained by means of differential ultraeentrifuga- animal for this agent of whatever type it may be. tion and found to be composed of small fragments Adenoviruses, for example, cannot be tested in of membranous vesicles, with adherent particles laboratory animals, and other technics have to identical to those of endoplasmic retieulum. Ribo- be employed for their diagnosis. There is an urgent nuelease acts specifically on the particles (338, need for identification of the particles observed 340) which are rich in ribonueleie acid and are in some eases of human leukemia and malignant connected with protein synthesis ('254, 414-416). lymphoma. The difficulties encountered in the Similar studies on the so-called microsomal frac- morphological studies of biopsy specimens are tion obtained from tumors, especially those of not insurmountable, and tissue culture specimens, viral etiology, are indeed necessary. Many animal combined with morphological and immunological viruses are composed of ribonucleie acid and pro-

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. 10(}~ ('aneer Heseareh Vo]. ~(), August 1960 tein. In animal viruses the central core, rich in (~80, ~81). Experhnents along these lines should RNA, varies in diameter from 50 to 4{)0 A and be exlenr and combined with the characteriza- is surrounder by a single or double membrane, tion of the nucleic acids involved. presumably a protein coat. The mierosomal par- Preparations made by means of the phenolic tieles which resemble the nueleoid of animal vi- deproteinization technic (~0~) from leukemic or- ruses have a diameter of 150-~00 A but do not gans of miee with spontaneous lymphatic leukemia have a surrounding membrane. Electron micro- and front mouse embryo cultures infected with seopic studies have so far failed to show any con- polyonm have been observed to induce nmltiple nection between these particles and the develop- tumors in suitable mice and hamsters, both on ment of any of the virus particles present in animal direct inoculation and following passage on nmuse or hulnan tulnors. embryo cultures, in which they induced cytopathic Some animal viruses, such as vaccinia and effect (139). Electron microscopic examination of adenoviruses (397), are known to contain DNA, the cells of mouse embryo cultures with cytopathic while other animal viruses contain RNA (s 185, changes following treatment with the nucleic acid 19~, 39~-394, 478). l{ous sarcoma virus contains preparations from polyoma-infected tissue culture RNA (~7, s as clearly shown by the application material has revealed the presence of characteristic of t)iochemieal anti electron microscopic methods particles in the nucleus of such cells (Fig. s (169). Highly purified preparations of RNA from similar to those observed in the cells of mouse tobacco mosaic virus induce the disease and form embryo cultures treated with polyoma prepara- the complete virus in tobacco leaves (18~2, 183, tions (139). Similar particles could not be found ~@2). The application of biochemical methods and ira the untreated mouse embryo cultures. Further electron microscopy has led to the localization experiments are needed to show a eonraection of the RNA in tobacco mosaic virus (184, s between the characteristic particles and the tumor- 467). Specific activity of I{NA has recently been inducing activity. Nevertheless, these experiments reported in a number of animal viruses: polio- open up a new possibility for the use of electron myelitis (7, 8, 95, 96, 3~24, 3~5), Semliki Forest, microscoI)y in the tests of preparations containing West Nile, Mengo, nmrine and equine encephalitis nucleic aci(Is from tissues infected with tumor (90, 96, 186), influenza (375), encephalomyoeardi- viruses. tis (~55), aim foot-and-mouth disease (78, 440). Electron microscopy has shown that the mor- It is of great interest that RNA from poliomyelitis phology of cells does not end at the micron scale, virus has been found to be infective for the cells but extends to the Angstroln and to the molecule. of normally resistant species grown in vitro and It has undoubtedly contributed to present-day to produce virus particles in these cells with anti- knowledge of cell structure by revealing an essen- genie i)roperties of the polio virus (~43, ~44, 3~4, tial similarity of the structure of cells of plants, 3~5). The I{NA from polio and other enteroviruses animals, and man. It. has further shown that has also been found infective for nonsuseeptible cellular" components have certain physiological and animals (~43, ~44). chemical functions. Thus, electron microscopy has Recent reports on the infectivity of RNA- led to a union of form and function, morphology containing preparations from a number of anilnal and biochemistry of cells, laying foundations for viruses (430) indicate that similar attempts should a much wider, deeper, and more extensive ap- be made to obtain active RNA anti DNA prepara- proach to the study of malignant cells and their tions from tumors of known and unknown viral constituents. Although electron microscopy, with origin. The recent progress in the purification of present-day fixation methods, allows for examina- llous sarcoma, erythroblastosis, and m veloblas- tion of thin sections of specimens at the ~0 A tosis viruses should provide at least partly purified or even 10 A level, at higher resolutions it is not virus material for studies of the activity of nucleic possible as yet to discern the molecular structure acid from such material. Indeed, the first steps of proteins, lipide membranes, anti nucleic acid. have already been made in this direction. The It appears, therefore, that new lnethods of fixatiou induction of mouse leukemia has been reported and processing of specimens for electron micros- with nucleic acid prel)arations froln organs of copy are urgently needed. It remains to be seen mice with spontaneous leukemia ('236) ancl from how much new structural detail will be obtained human leukemic lymph nodes (~25~5). Act'eleration 11)5" lhe recently introduced methods of staining of the apl)earance of leukemia and the induction with heavy metals (458, 459). Present-day electron of a variety of et)ithelial arr(t mesothelial tumors microscopy has shown that viruses are not mole- has been observed in nlice injected with nucleic cules, as at first considere(t when tobacco mosaic acid preparati()ns from mouse leukenaic organs virus was crystallized, but have a complicated

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. D~tOCHOWSKI--Electron Microscopy of l'iruses and Tumors 100~ structure like that of cells. It has helped to show Intracytoplasufic "Virus-like" Particles of Ehrlieh Ascites the basic constituents of specific activity of viruses Tmnor Cells. J. Biophys. & Bioehem. Cytol., 3: 161-70, 1!}57. and their localization within a virus particle. When ~. - ...... All Electron Microscopic Study of hwomplete one has learned that nucleic acids are the basis Virus Formatiou. Infection of Ehrlich Aseites Tumor of viral activity, the question arises whether or Ceils with "Chick Embryo-adapted" Newcastle Disease not the nucleic acids, especially deoxyribonucleic Virus (NI)V). J. Fxper. Mcd., 106:617-~6, 1957. acM, are responsible for the malignant change. 5. AFZELIUS, B. A. Tile Ultrastrueture of the Nuclear Mem- brane of tile Sea Urchin Ooeyte as Studied with the Elee- Electrossible to trace the

Intracelhtlar and Extracellular I)articles in Tissue Cul- 4~2. BI';ItNII.~.III), W.; BAUER, A.; GUI-'RIN, M.; and ()BERLIN(~., tures Inoculated with Parotid Tumor Agent (Polyoma C. f2tude au microscope 61ectroniquc dc corpuscles d'as- Virus). Proc. 17th Ann. Meeting Electron Microscope pect virusal dans des 6pith61iomas manunaires dc ]a Soe. Am., p. 17, 1959. souris. Bull. Cancer, 42:163-78, 1!155. "23. BANG, F. B. Morphology of Viruses. Ann. Rev. Micro- 43. BERNIiARD, W. ; BAUER, A.; tIAREL, J.; and ()BEltI.ING, biol., 9:'21-44, 1955. C. Les formes intracytoplasmiques du virus fil)romateux "24. B.txG, F. B.; AXDEI~VO~T, II. B.; and VELLlSTO, I. dc Shope. ]~tudes de coupes ultrafines au microscope Electron Microscopic Evidence Concerning the Mam- 61ectronique. Bull. Cancer, 41:4~8-44, 1955. mary Tumor Inciter (Virus). I [. An Electron Microscopic 44. BERNHARD, W.; Bo~.~a, R. A.; BEAJaD, D.: and BEARD, Study of Spontaimous and Induced Mammary Tumors J. W. Ultrastructmc of Viruses of Myeloblastosis and of Mice. Bull. Johns tlopkins IIosp., 98:~87-308,1956. Erythroblastosis Isolated from Plasma of I~ukcmic Chick- ~5. BANG, ~'. B.; VF,LLISTO, I.; and LIBERT, R. Electron ens. Proc. Soc. Exper. Biol. & Med., 97:48-5~2, 1958. Microscopic Evidence Concerning the Mammary Tumor 45. BERNHARD, W.; DONTCHEFF, A.; ()BERLIN-G, C.; and Inciter (Virus). I. A Study of Normal and Malignant VIGIER, P. Corpuscules d'aspect virusal dans los ccllules Cells from the Mammary Gland of Mice. Bull. Johns du sarcome de Rous. Bull. Cancer, 40:8l 1-~1, 1953. Hopkins IIosp., 98: ~55-85, 1956. 46. BERNHARD, W.; FEBVnE, H. L.; and CICAMEIr R. Mise ~ BA.IgRNETT, R. Z., and PALADE, G. E. Applications of en 6vidence au microscope 61ectronique d'un virus dans tlistochemistry to Electron Microscopy. J. Histochem. des cellules infect6cs in vitro par l'agent du Polyomc. & Cytoehem., 6:1-1~, 1958. Compt. rend. Acad. So., 249:483-85, 1959. "27. B.~TUFa~, R. The Nucleic Acid Content of Partially Purl- 47. BERNH.A_RD, ~[.; (]-AUTIER, A.; and ROI_YILLER, C. lJa tied Rous No. 1 Sarcoma Virus. Brit. J. Cancer, 11: notion de microsomes et le probl~me de la basophile 611-19, 1957. cytoplasmique; 6rude critique et exp6rinlentale, Arch. o8. -- .... . Relationship between Infectivity and the Ribo- Anat. Microscop. et Morphol. Exper., 43:~,36-75, 1954. mmleic Acid Content of Partially Purified Rous Sarcoma 48. BERNIIARD, W., and GRoss, I,. Pr6sence de particules Virus Preparations. Ibid., 12 :~56-68, 1958. d'aspect virusal dans les lissus tumoraux de souris at- f29. BEAMS, H. W.; TAtlMISIAN, T. N.; ANDERSON, E.; and teintes de leuc6mies induites. Compt. rend. Acad. Sc., DEWNr, R. L. The Structure of Nuclear Membrane in 248:160-63, 1959. Larval Gonads of Heliothis obsoleta. Proc. Soc. Expcr. 49. BERNHARD, W., arid (iu~.lux, M. Evaluation quantitative Biol. & Med., 91:478-75, 1956. du virus dans des tumeurs mammaires spontan6es au ~0. BEARD, J. W. Etiology of Avian Ieukosis. Ann. N.Y. grcff6es dc ditt'6rentes souches dc souris et 6tude de ses Acad. So., 68:478-86, 1957. rapports avec l'apparcil de Golgi. !d. Int. Syrup. on 31. B~AaD, J. W.; SUAm,, D. G.; and ECKErtT, E. A. Tumor ManImary Cancer, Pcrugia, pp. 6!7-40, 1957. Viruses. Adv. Virus Research, 3:149-97, 1955. 50.- .... Pr6sencc dc I)arliculcs d'aspect virusal, dans ;{~2. BEAUDIiEAC, Cr. S.; BECKF,R, C.; SIIARI', D. G.; PAINTER, les tissus tumoraux de souris atteintes (h" lcu('6mie spon- J. C.; and BEARD, J. W. Virus of Avian Myeloblastosis. tan6e. Conq)t. rend. Acad. So., 247:180~-5, 195S. XI. Release of the Virus by Mycloblasts in Tissue Culture. 51. BERNIIARD, W.; (~uI;ZIr M.; an(t ()BFRLING, C. Mist J. Nat. Cancer hist., 20:351-82, 1958. cn 6vidcnce dc corpuscules d'aspect virusal (tans diff6- ~{. BI'.'NEDETTI, I~. L. Pr6sence dc corpuscules identiqucs rcntcs sou('hes dc cancers mammaircs de la souris. Acta 'h ccux du virus de l'6rythrol)lastose aviairc chcz l'embry- Internat. contra cancrum, 12:544-47, 1956, ons du poulct ct los poussins normaux. Bull. Cancer, 5"2. BEI~Nm~ICD,W.; I|.~(~UEXA(r, F.; (}AVTIE1L A.; and ()BER- 44: 4478-8~2, ]957. LING, C. La structure submicroscol)ique des 616ments ~4. BENEDETTI, E. L., and BERNHARD, W. Pr6scnce de par- basophiles cytoi)lasmiques dans le f

oarticulibres observ6es au microscope 61ectronique dans HtrNTER, M. d. Agent in AK Leukemic Tissues not Sedi- certaines cellules leuc6miques. Compt. rend. Acad. Sc., mented at 105,000 X g Causing Neoplastic and Non- 240:459-60, 1955. neoplastic I~esions. Proc. Soc. Exper. Biol. & Med., 99: 61. ------. ~xamen au microscope 61ectronique des cellules 401-7, 1958. des leuc6mies myeloides. Bull. Microscop., 2:9-11, 1955. 81. BUNTING, II. Close-packed Array of Virus-like Particles 6~. . ~xamen des cellules leuc6miques au microscope within Cells of a Human Skin Papilloma. Proc. Soc. 61ectronique par la m6thode des coupes. Presse M6dicale, Exper. Biol. & Med., 84:3~7-39, 1953. 63:189-90, 1955. 8~. BURGOS, M. H., and FAWCETT, D. ~r. Studies on tile Fine 63. BESSlS, M., and THEIaY, J. P. Ultracentrifugation de leu- Structure of the Mammalian Testis. I. l)ifferentiation cocytes normaux et leuc6miques. ]~xamen au microscope of tile Spermatids in the Cat (Fells domestica). J. 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FiGs. 1, 2.--Api)earance of some cell constituents in benign cells of plaque, another benign precursor lesion of squamous- precursor lesions of "cancer eye" in cattle (141) 9 cell carcinoma of the eye in cattle 9Mag. X 14,000. FIG. 1.--Appearance of nucleus (N) and nucleolus (NU)in FIG. 3.--Visceral lymphomatosis of chickens (116, 118, 119, a cell of papilloma, a benign precursor lesion of squamous-cell 125). Cytoplasm (CY) of a cell from the affected spleen of carcinoma in cattle 9Mag. X9,000. chicken with visceral lymphomatosis. Inclusion bodies (IB) Fio. 2.--Appearance of nucleus (N) and nucleolus (NU) in with characteristic particles, vacuoles (V). Mag. X34,000.

455. WATSON, M. L. Pores in Mammalian Nuclear Membrane. 467. WILLIAMS, R. C. Electron Microscopic Studies of Tobacco Biochim. et Biophys. acta, 15:475-79, 1954. Mosaic Virus and of Nucleic Acids 9Spec. Publ. N.Y. 456. ------9The Nuclear Envelope. Its Structure and Rela- Acad. Sc., 5:~07-16, 1957. tion to Cytoplasmic Membranes. J. Biophys. & Biochem. 468. ---- 9The Role of Electron Microscope in Virus Re- Cytol., 1:~57-70, 1955. search. Int. Rev. Cytol., 6:1~9-91, 1957. 457. ------. Staining of Tissue Sections for Electron Micros- 469. WILLIAMS, R. C., and BACKt~S, R. C. The Electron Micro- copy with Heavy Metals. Ibid., 4:475-78, 1958. graphic Structure of Shadow-cast Films and Surfaces 9 458. ------9Staining of Tissue Sections for Electron Micros- J. Appl. Physics, 20:98-106, 1949. copy with Heavy Metals. II. Application of Solutions 470. WILLIAMS, R. C., and WYCKOFF, R. W. G. The Thickness Containing Lead and Barium. Ibid., pp. 7~7-30. of Electron Microscopic Objects. J. Appl. Physics, 15: 459. ------9Further Observations on the Nuclear Envelope 71~-16, 1944. of the Animal Cell. Ibid., 6:147-56, 1959. 471. ----- 9Applications of Metal Shadow-casting to Electron 460. WEISS, J. M. The Ergastoplasm. Its Fine Structure Microscopy. Ibid., 17: ~3-33, 1946. and Relation to Protein Synthesis as Studied with the 47~. ~rITTER, R. F.; WATSON, M. L.; and COTTONE, M. A. Electron Microscope in the Pancreas of the Swiss Albino Morphology and ATP-ase of Isolated Mitochondria. J. Mouse. J. Exper. Med., 98:607-18, 1953. Biophys. & Biochem. Cytol., 1:1~7-38, 1955. 461. ------. The Role of the Golgi Complex in Fat Absorption 473. WOOLLEY, G. W., and SMALL, C. Experiments on Cell-free as Studied with the Electron Microscope with Observa- Transmission of Mouse Leukemia. Cancer, 9:110~-6, tions on the Cytology of Duodenal Absorptive Celts. 1956. Ibid., 102: 775-81, 1955 9 474. WYCKOFF, R. W. G. Electron Microscopy. New York: 46~. ------9Mitochondrial Changes Induced by Potassium Intersciencc Publications, 1949. and Sodium in the Duodenal Absorptivc Cell as Studied with the Electron Microscope. Ibid., pp. 783-88. 475. YAsvzt~MI, G., and SUGIHARA, R. A Comparative Elec- tron Microscopic Study on Ehrlich Ascites Tumor Cells, 463. ------9Intracellular Changes Due to Neutral Red as revealed in the Pancreas and Kidney of the Mouse by Yoshida Sarcoma Ceils, and Human Peritonitis Ascites Electron Microscope. J. Exper. Med., 101:~13-24, 1955. Cells 9Cancer Research, 18:1167-70, 1958. 464. WEISSENFELS, N. Dcr Feinbau und das Verhalten dcr 476. YASUZUMI, G.; SUGIHARA, R.; KIRIYAMA, M.; IKEDA, Mitochondricn in Tumorzellen. Krebsforsch. Krebsbe- T.; and HmASRIZAWA, S. Electron Microscope Studies k~impfung, 9. : 10~-8, 1957. of Sections of the Ehrlich Ascites Tumor. I. Ultrastructure 465. WERNER, G. H., and SCHLESINGER, R. W. Morphological of Cytoplasm of Ehrlich Ascites Tumor Cells. J. Nara and Quantitative Comparison between Infectious and M. Assoc., 7:135-38, 1956. Non-infectious Forms of Influenza Virus. J. Exper. Med., 477. ZETTERQVIST, H. The Ultrastructural Organization of the 100: ~03-16, 1954. Columnar Epithelial Cells of the Mouse Intestine 9Stock- 466. WESSEL, W., and BERNHARD, W. Vergleichende elektro- holm, 1956. nenmikroscopische Untersuchung yon Ehrlich-und Yoshi- 478. ZILLIG, W., SCHAFER, W.; and ULLMAN, S. Ueber den da-Ascitestumorenzellen. Ztschr. Krebsforsch., 62:140-- Aufbau des Virus: Elemcntarteilchems bet klassischen 6~, 1957. Gefltiegelpest. Ztschr. Naturforsch., 10B: 199, 1955.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. Fins. 4-7.--Granuloblastosis (myeloblastosis) in chickens (118, 1~0-1~, 1~6). FIG. -~.--Part of the cytoplasm (CY) of a cell from spleen of chicken with granuloblastosis, showing inclusion bodies (IB) with characteristic particles, vacuoles (V), and membranous structures in the upper left-hand corner. Mag. XS~,000. Fro. 5.--Part of tile cytoplasm (CY) of another cell with mitochondria (M) iu vorious stages of alterations. )'lag. X37,000. Fro. 6.---Inclusion bodies (IB) in ttle cytoplasm (CY) of another (,ell with numerous characteristic particles. Mag. X 63,000.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. FIG. 7.--General appearance of (:ells from the affected splee[~ of chicken wiCh granuloI~lastosis. Mag. )K9,000. FIGS. 8-11.--Erythroblastosis of chickens (120-1~4). FIG. 8.--General at~pearance of cells ~rom lhe affected spleen of chicken with erythroblastosis strain RPL-I~2. Mag. )KG,000.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. FIG. 9.--An inclusion body with charat'teristie partMes ill the eyloplasm of a cell from spleen of chicken with erythroblastosis strain RPL-I~. Ma~. X 110,000. Fro. 10.--Part of the cytoplasm with memt~ranous structures (MS) and characteristic particles in inclusion bodies (IB) undergoing vammlization (V). Erythroblastosis strain RPL- 1~. Mag. X6S,(}I)0. Fro. 11. Part of cytoplasm (('1") of cells from spleen of chicken with erythroblastosis strain R. Melnbranous structures (MS), mitoehondria (M), inclusion bodies (1B) with characteristic particles. Mag. X:iv),O00.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. Fla. lg.--Adenoearcinomz of the kidney of chiekens induced l~y myeloblastosis virus (135). Appearance of tile eharaeteristie particles. Mag. X54,000. FIGS. 1S-16.--Mammary tumors of mice (133, 18-~). Fro. 13.--Appearance of upper zone of a high-speed centrifugal pellet from defatted and deeaseinated agent-carrying mouse milk with eharaeteristie particles. Mag. XS0,000. Fro. 14.--Similar particles in a pellet from milk of mice of another agent-earrying strain. Mag. X 130,000. Fro. 15.--Similar particles following fluorocarbon treat- merit of a high-speed centrifugal pellet from agent-carrying mouse milk. Mag. X60,000. FIG. 16.--Appearance of eharaeteristie particles in a spontaneous mouse mammary tumor. Mag. X70,000.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. FrGs. 17-~0.--Mouse leukemia (115-117: 130). Fro. 17.--Part of tile cytoplasm (CI') of a cell from the thymus gland in spontaneous lymphatic leukemia of mice. Membranous structures (MS), osmiophilie bodies (OB). Mag. X 16,000. FIG. 18.--Cllaracteristic partMes in the interce]lular space of a cervical lymph node of a mouse with lymphatic leukemia. Mag. X ~4,000.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. Fro. 19.--Part of the eytoplasm and nuclei of cells from a cervical lymph node of a leukemic mouse. Characteristic partieles may be seen. Mag. X43,000. FIG. 30--Appearance of tile characteristic particles ill spontaneous and induced lymphatic leukemia of mice. Mag. X64,000. Fins. 31-38.--Parotid gland tumors of mice (117, 137, 139). FIG. 31.--Appearance of eharaeteristie particles in a parotid gland tumor indueed in a mouse by a cell-free preparation from leukemic organs in spontaneous mouse leukemia. Mag. X63,000. Insert: A partiele at. higher magnification. X96,000. FIG. ~L--Characteristic particles in an inclusion body (IB) present in the cytoplasm (C}') of a parotid gland tumor. Mag. X 54,0O0.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. FtG. ~3. -General appearance of "l parotM gland tumor eel[ showin~ nucleus and cytoplasm with welt developed endoplasmic retieulum. Mag. XI$,O00. Fins. ~, ~6, ~7, ~8.- Polyoma virus (131, 18~). Fw,. ~I,. I'art of the cytoplasm (('1") and nucleus (N) of a mouse e:,nhryo cell grown in ritr~l a,d infected with nolyoma. Numerous particles present in the nucleus. Mag. X3~,000. FiG. q5.-- Ai)l)earanee ()f part of the cytopl'tsm (('1") ()f a cell from a lymph node of a mouse with x-ray-induced leukemia ()smi,)philie bodies I OBi, vacuoles (.V), mitoel,ondria (.11). Mag. X3~,000.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. F~(~. ~6.--Charaeteristic appearance of many nuclei of'cells from kidney of a mouse infected with polyoma. Nuclei (N) with charaeteristie particles, cytoplasm (CY) with mitochondria (M). Mag. X~21,000. Fro. ~27.--Characterisfic particles in the nucleus (N) of a mouse embryo cell infected with polyoma. Nuclear membrane (NM), cytoplasm (CY). Mag. XT~,000. FIG. ~8.--Appearance of a nucleus (N) of mouse embryo cell grown in vitro and treated with the aqueous phase of phenolic extract of polyoma-infected mouse embryo culture material. Mag. X3~,000. Fins. y29, 80.---X-ray-indueed leukemia in mice (116, 117, l~S). See also Fig. ~5. FIG. ~9.--Part of the cytoplasm of a cell from a lymph node of a mouse with leukemia induced by cell-free preparation of leukemic organs of mice with x-ray-induced leukemia. Inclu- sion body (IB) with characteristic particles, mitochondrion (31). Mag. X44,000. Fro. 80.--Another part of the cytoplasm o~ the same cell as in Fig. gg, showing vacuoles (V) with vharaeteristic particles (P). Mag. X67,000.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1960 American Association for Cancer Research. Fins. 31 34.-]Imnan leukemia (116, 117, 136, 1~0). Fro. 31.- (;eneral appearance of tile cytoplasm ((')') of cells from a lymph node of a patient with acute lymphatic leukemia. Some mwlei may be seen, cytoplasm ((;1") showing vacuoles (1") and altered mitoc]ton(h'ia (M). Mag. X5,(~)O. FIG. 3~.--Parl of the cytoplasm (Cl') of a <,ell from lymph node of a t)atie,~t with malig,,ant lymplioma, hwh,sion bodies (IB) with ehara(,tcristie pavtMes. Mag. X38,000. Ft(;. 33.--Part of the eytot)lasm (CI') of another cell from th(" same ease of malignant lymphoma. Charaeteristio partit:les {I)). Mag. X37,0[)0. FI(]. 34.--An inclusion h()dy witll cha,'actc,'istic I)a,'ti(:h's in ,ells grown ft~ rfiro from the same ease of malignant lymphoma. Mq~. X~4,000.

Leon Dmochowski

Cancer Res 1960;20:977.

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