key: cord-0009842-23cdi61w authors: Györkey, Ferenc; Sinkovics, Joseph G.; Györkey, Phyllis title: Electron microscopic observations on structures resembling myxovirus in human sarcomas date: 2006-06-27 journal: Cancer DOI: 10.1002/1097-0142(197106)27:6<1449::aid-cncr2820270627>3.0.co;2-3 sha: 11c686b3de7f984cb5e82860014a23050d7cf740 doc_id: 9842 cord_uid: 23cdi61w Human tumors of mesenchymal origin contain cytoplasmic structures resembling ribonucleoprotein strands of paramyxoviruses. Similar structures have previously been reported in collagen diseases. The nature and function of these structures remain unresolved. H u m a n tumors of mesenchymal origin contain cytoplasmic structures resembling ribonucleoprotein strands of paramyxoviruses. Similar structures have previously been reported in collagen diseases. The nature and function of these structures remain unresolved. T duced tumors of animals and naturally occurring tumors of man lacks valid criteria. One discrepancy concerns the viral etiology of animal and human malignant neoplasms. In certain animal tumors, such as murine leukemia, sarcoma, and mammary carcinoma, RNA-containing type C and type B virus particles occur in large numbers. Bioassays have irrefutably proved that these virus particles are the causative agents of these tumors.7821. 27,32 I n some other types of experimental tumors, no mature virions appear but there is oncogenic viral DNA incorporated into the genome of the host cell.9 Not only DNA-but also RNA-containing viruses may exist in the neoplastic cells in the form of subviral structures.lG Cell-fusion, heterokaryon formation, and "rescue" by a helper virus may be needed for the maturation of the subviral structures into complete ~irions.16.28~29 Most human tumors when examined in the electron microscope or tested in bioassays appear to be devoid of mature virions. Extensive search is required to find a few elusive viruslike particles in human tumors. Herpes, adeno-and reoviruses, as well as elementary bodies of mycoplasma, occasionally occur in human tumors probably as passengers and not -~ as etiologically important agents.1~8~20 Occasionally, type C and related virus-like particles were found in neoplastic tissues deriving from human leukemia, lymphoma, and sarcoma.8J6 T h e number of these particles rapidly diminishes in cells cultured in vitro; thus, these viruslike particles have never been identified as the human counterparts of oncogenic type C viruses of animals. On the contrary, culturing in vitro, particularly of lymphoid cells deriving from human lymphoma and from other lymphoproliferative entities, results in the rapid increase in the amount of a herpes-type ( E p stein-Barr) virus. 22 Very little attention has been paid in the past to the existence of subviral structures in tumor cells of man. Preliminary findings concerning such structures, however, have recently been reported by Stewart36 who observed the occurrence of filamentous structures and budding type Clike virus particles in tissue cultures of human liposarcoma and Hodgkin's disease. I n the latter case, these structures were identified later as ribonucleoprotein strands of simian paramyxovirus 5. 37 Lymphoid cells grown in culture from patients with infectious mononucleosis4 or from idiopathic thrombocytopenic purpura32 occasionally contain lattice-like arrangements of short strands which, in cross section, appear as aggregates of small virus-like particles. The viral nature of these latter structures have been questioned; the structures were presented also as cellular elements.2. 3 Thus, merely morphological studies failed to determine in most instances whether the filamentous structures represented viral nucleoprotein strands or cellular material. If the structures are of viral derivation, it remains CANCER Julie 1971 VOI. 27 unresolved whether these structures are components of a passenger virus or of a virus etiologically related to the induction of mesenchymal tumors of man. Thus, the need for a complex study has become evident. Such a study should deal with the occurrence and morphological features of these hypothetically subviral structures, and it should encompass attempts at isolation and identification of these structures as well as bioassays to establish whether the structures possess any oncogenic potency or not. T h e present paper is a preliminary report on morphological observations concerning structures that may be of viral derivation, as found in biopsies and tissue cultures of human tumors of mesenchymal origin. Specimens of tumors: Twelve tumor biopsies (3 rhabdomyosarcomas, 3 liposarcomas, 2 chondrosarcomas, 1 osteosarcoma, 1 fibrosarcoma, 1 neurofibrosarcoma, and 1 Kaposi's sarcoma) and 12 primary tissue cultures O€ tumors (8 rhabdomyosarcomas, 2 chondrosarcomas, 1 fibrosarcoma, and 1 osteosarcoma) were examined electron microscopically. Electron microJcopy: Finely minced freshly biopsied tumor tissue or cultured cells dispersed by trypsinization or scraping were placed immediately in glutaralclehyde, postfixed in osmic acid, dehydrated in graded series of ethanol, embedded in Epon Araldite, cut with an LKB ultratome, stained with ur-any1 acetate followed by lead citrate, and examined in an RCA 3G electron microscope. Tissue cziZtures: Finely minced fresh tumor biopsy material was placed in glass T flasks under perforated cellophane membrane antl incubated in Ham's FIO medium containing 20% heat inactivated fetal calf serum, penicillin G ?00u/ml, streptomycin 100 pg/nil, antl Lanamycin 115 pg/ml. Cultured cells were T h e discovery of the structures required extensive search; several sections of each specimens and several liiintlretls of fields were examined. T h e distribution of the structures was found to be quite irregular; in some sections, it was easy to find the structures, whereas other sections of the same specimen were devoid of them. Small clusters of mature virions were seen only exceptionally in the specimens examined (Fig. 1) ; these findings will be reported elsewhere after lurtlier studies. Normal kidney, skin, and leukocytes were examined for the presence of similar structures. Extensive search involving a total of 50 tissue specimens yielded negative results. Morphological description of the stnrctures: T h e filamentous and tubular structures are located in the cytoplasm in aggregates of varying sizes. T h e aggregates may be membranebound. In the case of the Kaposi's sarcoma, the structures were found between the nuclear membranes. T h e filaments measure 200-220 A in diameter and u p to 1000 A in length. In cross sections, the curved filaments appear as both electron-dense or lucent round bodies (Figs. 2-4) . Further studies are needed to determine the nature and significance of these structures. It is uncertain whether the structures as found in different tumors are of the same type. While some of these structures closely resemble unenveloped ribonucleoprotein strands of paramyxovir~i~es.~~ other slightly different, i.e., smaller, structures, particularly those appear-ing in cultured lymphoblasts,'s32 are thought to represent cellular material.2JaG During the replication of parainfluenza virus type 2 in HeLa cells, cytoplasmic areas containing a dense network of filamentous structures without mature virions were seen.19 I t was postulated that the viral capsid is acquired when the viral nucleoprotein strands are assembled beneath the cell membrane and the virus particles pass through the cell membrane by a budding process.*Q I t is compatible with this view that the filamentous structures described in this report occur at cytoplasmic sites where replication of viral nucleoprotein takes place. Myxoviruses and type C oncoRNA viruses display morphological resemblances, thus some of the structures described herein may represent ribonucleoprotein strands of a human oncogenic virus of the type C or related class. Type C viruses have recently been incriminated in the causation of human sarcomas.11323 In tumors caused by Rous sarcoma virus in primates, crystalline and filamentous cytoplasmic structures somewhat similar to those reported i n this paper have been found.24 T h e occurrence of quite similar structures in collagen diseases of man, particularly, as first shown by us, in systemic lupus erythematosus,13817,1*,33 polymyositis,5~'0 Sjogren's syndrome,:{o and discoid lupus,14 indicates that these structures are not confined solely to tumors. T h e ribonucleoprotein composition of tlie filmientous strands found in lupus was shown by digestion with RNAase.12 Isolation of vii '11 agents failed from biopsy materials containing the filamentous structures.10 Thus, it was aljo proposed that similar strands represented cellular and not viral elements."SJ'.25 Even if these structures are of viral derivation, a5 we ha proposed,13131*3a it remains to be determiiied whether they play the role of a passenger or of an initiator in collagen diseases arid in mesenchymal tumors. Members a f the orthomyxo-and paramyxovirus groups may elicit or aggravate "autoimmune" reactions by endowing the host cells, in which viral components replicate with "neoantigenic" features; thus, subviral structures may be of great etiological significance in collagen diseases.34 Paraniyxovirus and coronavirus-like structures have recently been found associated with viral hepatitis,l5,3*,39 but it remains to be determined whether these structures play the etiologic role or act only as passengers. Autoimmune sequelae of viral hepatitis are well known; tlie harboring of subviral structures within parenchymal liver cells could explain snch "autoimmune" reactions. T h e structures described in this paper may represent unenvelopecl ribonucleoprotein strands of an ubiquitous virus which is widely spread in human populations and seldom reaches full maturity. It is evident that further morphological observations, in addition to virologic, serologic, and biologic studies, are needed in order to elucidate the significance of these filamentous structures in mesenchy-ma1 tumors of man. Further isolations of reovirus type 3 from cases of Burkitt's lymphoma. Brit. Alerl. J . 2. Chandra, S.: tlndulating tubules associated with endoplasmic reticullnu in pathologic tissue. Lab. I nvest Similarity between leukocyte cultures from cancerous and noncancerous human subjects: a n electron niicroscopic study T h e circulating lymphocyte. Its role in infectious mononucleosis Myxovirus-like structures accompanying nuclear changes in chronic polymyositis Tubular structures associated with the endothelial endoplasmic reticulum in glomerular capillaries of Rhesus monkey and nephritic man T h e mammary tumor system in mice, a brief review. I n Viruses Inducing Cancer Viral studies in human leukemia and lymphoma. I n Proceedings of International Conference on Leukemia-Lymphoma Viral isolation studies of inclusion positive biopsy from human connective tissue diseases Pathogenic effects of extracts of human osteosarcomas i n hamsters and mice Systemic lupus erythernatosus antl myxovi-1' 11s Discoid Iupns crgthematosus. Electron microscopic studies of paramyxovirns-like structures Coronaviiuscs and viral hepatitis Oncogenes of RNA tumor viruses a s determinants of cancer Cytoplasmic tubular sturctures in kidney biopics in systemic lupns erythematosus. Arthritis Xlreuin Virus-like stuctures in lupus erythematosus Electron mi-cro5copic examination of cytoplasniic inclusion bodies in cells infected with parainflnenza virus type 2. Viral-20 Tumors intlnced in juvenile and adult primates by chicken sarcoma virus Evidence for the in vitro transfer of defective Rons sarcoma virus genome from hamster tumor cells to chick cells Virus-like structures in Sjogren's syndroms. I.nncet 1:568 Working hypothesis: viral etiology of autoimmune diseases A comparison of murine and human leukemia A rapidly fatal case o'f systemic lupus erytheniatosus: structnres resemhling viral nncleoprotein strands in the kidney and activities of lymphocytes i n culture Leukemogcnesis stemming from autoimmune disease. I n Genetic Concepts and Neoplasia, T h e Twenty-third Annual Symposium on Fundamental Cancer Research, 1969, T h e LJniversity of Texas M. D. Anderson Hospital and l'nmor Institute at Houston Search for viruses in childhood leukemia and rhabdomyosarcoma. I n Leukemia in Animals antl Man T h e isolation of myxo type virus from two h u n~a n sarcomas Viruses in human tumors. I. Hodgkin's disease Virus like particles in liver disease patients' sera. Presentation a t the International Congress of Infectious Diseases Presence of particles other than the Australia-SH antigen in a case of chronic active hepatitis with cirrhosis