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Virus-Like Particles in 224Ra-Induced Murine Osteosarcomas
Beitr. Path. Bd. 152, 116-126 (1974)
Department of General and Experimental Pathology (Head of Dept.: Prof. Dr. W. GassInstitute of Biology, Gesellschaft fur Strahlen- und Umweltforschung mbH Munchen
NER),
Virus-Like Particles in O steosarcomas * **
224
Ra-Induced Murine
Virusahnliche Partikel in Ra-224-induzierten Osteosarkomen der Maus K.-H. MARQUART With 5 Figures' Received November
I,
1973 . Accepted February
II,
1974
Summary Electron microscopic investigation of osteosarcomas from three mice, induced by the incorporation of 224Ra, and an osteosarcoma metastasis from the liver of one of the animals revealed the presence of peculiar intracellular particles in all of the tumours. The particles were spherical, measured about 80 nm in diameter and consisted of two electron-dense, ring-shaped, membrane-like shells which concentrically surrounded an electron-lucent core. Nearly all of the particles with this doughnut-like ultrastructural appearance occurred in cisternae of the rough-surfaced endoplasmic reticulum (RER) of tumour cells. They were seldom observed in intracytoplasmic smooth-surfaced vacuoles or located freely in the cytoplasmic matrix. Crescent-shaped particles were occasionally found presumably in the stage of formation by budding into cisternae of the RER from its membrane component. The ultrastructure, size and formation of the particles, as well as their location in relation to intracellular structures, are similar to those described for type A viral particles. The significance of the virus-like particles, especially in relation to the pathogenesis of the malignant bene tumours, is not known at present.
Malignant bone tumours arise in mice as late effects following the incorporation of bone-seeking radionuclides such as 905r, 226Ra, 224Ra or 227Th (FINKEL and BISKIS, 1959, 1968, 1969; VAN PUTTEN and DE VRIES, 1962; FINKEL * Dedicated to Prof. Dr. O. HUG on the occasion of his 60th birthday. ~ ..
Performed within the framework of an association contract EURA TOM-GSF (Con-
tract No. 090-72-1 BIAD).
Virus-Like Particles in Osteosarcomas . 117
et aI., 1969; HUG et aI., 1969; NILSSON, 1970; GaSSNER et aI., 1970, 1971 a, b). It is still not known whether the radiation, emitted from the incorporated radionuclides, induces osteosarcomas directly by possible biochemical lesions or indirectly by suppressing the immune response and unmasking a latent oncogenic virus infection. Since experiments have shown that malignant bone tumours can be induced in mice by the inoculation of viruses (STEWART et aI., 1958; SJOGREN and RINGERTZ, 1962; FINKEL et aI., 1966; YUMOTO et aI., 1970) or by immunosuppression (GAUGAS et aI., 1969; SIMPSON and NEHLS EN, 1971), the hypothesis of the viral origin of radiation-induced murine osteosarcomas has received greater attention. FINKEL and BISKIS (1969) have seen, in ultrathin sections of 90Sr bone tumours from mic~, particles which were similar in appearance to viral particles found in virus-induced murine bone tumours. Recently, membraneous structures resembling viral particles have been observed in primary or passaged osteosarcomas of mice, induced by the incorporation of 226Ra or 90Sr as well as by the inoculation of an oncogenic RNA virus (LLOYD and MACKEVICIUS, 1971). The present study describes the occurrence of virus-like particles in osteosarcomas from three mice, induced by the incorporation of 224Ra, and a metastatic tumour from the liver of one of the animals.
Materials and Methods The three mice belonged to a dose-protraction experiment carried out with the bone tumour-inducing 224Ra (Luz et aI., 1972). The ultrastructure of 224Ra-induced murine osteosarcomas was studied within the framework of this experiment. The present study is a preliminary report describing a finding which appeared to be of sufficient interest to be presented at this time. Four-week-old, female NMRI mice (Neuherberg strain) were injected, intraperitoneally, with 224Ra. 224Ra is a short-lived (half-life 3.64 days), alpha-emitting radionuclide. It is a bone volume-seeker which mainly deposits after injection into mice in sites similar to those in which calcium is deposited. Two mice of the present study received 12 ftCi/kg of body weight 224Ra protracted over a period of 4 weeks (the individual amounts of activity were 1.5 ftCi/kg each 3.5 days). One mouse was injected with 36 ftCi/kg of body weight 224Ra also protracted over 4 weeks (the individual amounts of activity were 4.5 ftCi/kg each 3.5 days). The activity of 12 ftCi/kg 224Ra, injected into a mouse, produces after total decay an accumulated average skeletal dose of about 360 rads, the activity of 36 ftCi/kg 224Ra one of about 1080 rads (MULLER,
197 1). 224Ra (Thorium X) was used as commercially supplied (Amersham Buchler GmbH & Co. KG, Braunschweig, Germany) in the form of a radium chloride solution containing 2-6.2 mg/ml calcium chloride. The solution contained impurities consisting of the long-lived radionuclides 228Th, 226Ra and 210Pb whose total activities were not more than 5 X 10-6 Ci per Ci 224Ra (MULLER, 1971). Before administration to the animals, the radium chloride solution was diluted with physiological saline to obtain the different amounts of 224Ra.
II
8 . K.-H.
MARQUART
The three injected mice were maintained on a standard pellet diet together with drinking water ad libitum. Eighteen months following the first 224Ra injection a protruberant tumour had developed in the left pelvic region of one animal which had been injected with 12 !lei/kg 224Ra. Autopsy of the mouse, after sacrifice using chloroform, and light microscopic studies revealed an osteoblastic osteosarcoma of the proximal part of the left femur and metastatic tumours in the liver. In the other mouse which had received 12 !lCi/kg 224Ra a tumour was recognizable in the medial pelvic region 19 months following the first incorporation of 224Ra. Autopsy of the animal showed an osteoblastic osteosarcoma of the os sacrum. Twenty months after the first 224Ra injection a prot rube rant tumor was found in the right lower head region of the animal which had been injected with 36 !lCi/kg 224Ra. Autopsy of the mouse revealed a fibroblastic osteosarcoma of the mandibula. This type of osteosarcoma is typical for 224Ra-induced murine osteosarcomas of the head region which scarcely show osteoblastic differentiation as do bone tumours of other skeletal sites (GassNER et a!., 1972). Neither leukemia nor any other pathologic alterations were found in the three tumour-bearing mice. Immediately after sacrifice of the mice, small pieces of tumour tissues, from the three osteosarcomas and one liver metastasis, were removed and placed in cold phosphate-buffered 6.25% glutaraldehyde (pH 7.3) or in the case of the tissue from the fibroblastic osteosarcoma of the mandibula in cold phosphate-buffered 1% osmium tetroxide (pH 7.3). Immersed in the fixative solutions the specimens were cut into cubes of about I mm 3. The tissue cubes were fixed in the glutaraldehyde for 4 hours followed by postfixation in cold phosphate-buffered 10f0 osmium tetroxide for 2 hours. The tissue cubes from the tumour of the lower jaw were primarily fixed in the osmium tetroxide for 2 hours. Following dehydration through a graded series of alcohols to propylene oxide, the specimens were embedded in Epon 8 I2. Sections, cut with a diamond knife on a Reichert Om U2 microtome, were stained with uranyl acetate and lead citrate and examined in an AEI EM 6B electron microscope.
Results Electron microscopic investigation of the tumour tissues from the three 224Ra-induced murine osteosarcomas and the liver metastasis revealed the presence of peculiar intracellular particles which were only visible at a magnification higher than I : 15,000. The occurrence of these structures in the different tumours varied slightly. Osteoblastic osteosarcoma of the femur. Many of the osteoblast-like cells from the osteosarcoma of the femur contained electron-dense, spherical particles measuring about 80 nm in diameter (Fig. I). The particles usually occurred singly (Fig. I, 2), but in a few cells groups of them were present. Higher magnification revealed that the particles consisted of two electron-dense, ring-shaped, membrane-like shells which concentrically surrounded a relatively electron-lucent core measuring approximately 45 nm in diameter (Fig. 2, 3). The inner membrane-like shell bounded the electron-lucent core and the outer one limited the particles. Occasionally, the inner shell
Virus-Like Particles in Osteosarcomas .
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Fig. r. A low magnification electron micrograph showing portions of two osteoblast-like cells from a 224Ra-induced murine osteosarcoma of the femur. In one cell a single electrondense, spherical particle (arrow) can be detected in a cisterna of the rough-surfaced endoplasmic reticulum. B, mineralized bone matrix. X 16,500.
appeared very electron-dense whereas the outer one was less clearly defined (Fig. 3). Nearly all of the particles with this so-called "doughnut-like" ultrastructural appearance were present in cisternae of the rough-surfaced endoplasmic reticulum (RER) of osteosarcoma cells (Fig. I-3). They were seldom observed free in the cytoplasmic matrix or in intracytoplasmic membranebound vacuoles (Fig. 3). In very few cells, the doughnut-like particles could also be detected within the perinuclear space. None of the particles were observed in intercellular spaces of the osteosarcoma tissue.
I20 .
K.-H. MARQUART
Fig. 2. Two doughnut-shaped particles (arrows) in cisternae of the RER of an osteosarcoma cell at higher magnification . Their electron-lucent core is clearly visible. X 60,000.
Crescent-shaped particles were occasionally found presumably in the stage of formation by budding into cisternae of the RER from its membrane component (Fig. 3). Various stages in this budding process were seen. Particles developed only from membranes of the RER. Other cellular membranes such as those limiting cytoplasmic vacuoles or the limiting cell membrane were never involved in this process. Osteosarcoma metastasis 0/ the liver. Doughnut-like particles, identical to those from the osteosarcoma of the femur, were also found in osteoblast-like cells from the metastatic tumour in the liver of the same animal. The particles were seen only rarely in cells from this tumour, and then in cisternae of the RER and in a few cases within the cytoplasmic matrix. No particles were observed in intracytoplasmic vacuoles and none were found budding into cisternae of the RER. Doughnut-shaped particles were never detected in intercellular spaces of the tissue from the liver metastasis. In cells of the liver tissue, adjacent to the metastatic tumour, no such particles were observed.
Virus-Like Particles in Osteosarcomas .
121
Fig. 3. A doughnut-like particle (arrow) can be observed in an intracytoplasmic mcmbranebound vacuole. Another particle (arrowhead) is located in a cisterna of the RER. A third particle (double arrow) is found budding into the RER from its membrane component. d, dense body. X 8o,oco.
Osteoblastic osteosarcoma of the os sacrum. Electron microscopic investigation of tissue from the osteosarcoma of the os sacrum also revealed the presence of intracellular doughnut-shaped particles identical in their ultrastructure to those described above. However in this tumour, relatively few of the particles were present. They occurred only singly in cisternae of the RER (Fig. 4) and seldom freely in the cytoplasm of a few osteoblast-like cells. No budding particles were seen and none of the particles were found in extracellular spaces of the tissue. Fibroblastic osteosarcoma of the mandibula. Doughnut-shaped particles, identical in structure and size to those which occurred in the tumours mentioned above, were detected in many of the cells from the osteosarcoma
122 •
K.-H.
MARQUART
Fig. 4. A part of an osteoblast-like cell from a 224Ra-induced murine osteosarcoma of the os sacrum is shown. A doughnut-shaped particle (arrow) can be observed in a cisterna of the RER. Trabecular mineralized bone matrix (B) is closely apposed to the cellular border. X 60,000.
of the mandibula. The particles were as frequent in this tumour as in the osteoblastic osteosarcoma of the femur. They occurred mainly in cisternae of the RER (Fig. 5) of tumour cells. Very few particles were located freely in the cytoplasmic matrix and a single particle was found in a smoothsurfaced vacuole of a Golgi complex. Crescent-shaped particles in process of budding into cisternae of the RER from its membrane component were frequently seen (Fig. 5). Ribosomes were absent from the membranes of the RER on sites of particle formation. Occasionally, the cisternal membranes from which particles developed were abnormally thickened (Fig. 5). None of the particles occurred in extracellular spaces of the tumour tissue. The average outer diameter of all particles, found in the three 224Ra_ induced murine osteosarcomas and the liver metastasis, was 80 nm, ranging from 70 to 90 nm. The average diameter of the electron-lucent core of the particles was 45 nm, ranging from 40 to 55 nm.
Virus-Like Particles in Osteosarcomas .
12 3
Fig. 5. Several doughnut-like particles are located in cisternae of the RER of a cell from a 224Ra-induced murine ostecsarcoma of the mandibula. One particle (arrow) appears to bud from a membraneous stalk into a cisterna of the RER. The membrane of this cisterna is abnormally thickened and lacks typically aligned ribosomes. X 80,000.
Discussion The particles, found in ultrathin sections of three 224Ra-induced murine osteosarcomas and a liver metastasis, show the following morphological characteristics: 1. The particles occur only intracellularly. 2. They are spherical and consist of two electron-dense, ring-shaped, membrane-like shells which concentrically surround an electron-lucent core (doughnut-like ultrastructural appearance). 3. The average diameter of the particles is 80 nm, that of the electron-lucent core 45 nm. 4. Nearly all of the particles occur in cisternae of the RER of tumour cells. They are seldom observed in smoothsurfaced vacuoles or located freely in the cytoplasmic matrix. 5. Crescentshaped particles are occasionally found presumably in the stage of formation by budding into cisternae of the RER from its membrane component. The ultrastructure, size and formation of the particles, as well as their location in relation to intracellular structures, are similar to those described for type A viral particles (BERNHARD, 1958; BERNHARD and GUERIN, 1958;
Il4 .
K-H.
MARQUART
DALTON, 1962, 1972). Following the sub-classification of DALTON (1962) the particles resemble type As viral particles. The occurrence of type A particles in radiation-induced osteogenic sarcomas has not yet been reported. Such viral particles have been described in cells of various other mouse neoplasms such as mammary carcinomas, Ehrlich ascites tumours, melanomas, leukemias, plasma-cell tumours, salivary gland tumours, precancerous hyperplastic mammary tissues, lymphomas and mastocytomas (for references see BERNHARD, 1958, 1960; BERNHARD and GROSS, 1959; DALTON et aI., 1961; PEARSON and BAKER, 1961; DALTON, 1962; DE HARVEN, 1968). The virus-like particles which have been revealed by FINKEL and BrSKrs (1969) in osteosarcomas induced in CFI, CBA and X/Gf mice by the incorporation of 90Sr are different in their ultrastructural appearance from those reported in the present study. In contrast to the observed occurrence of viruslike particles only within cells of 224Ra bone tumours, these authors saw doughnut-like particles, consisting of three concentric membrane-like shells, in extracellular spaces as well as in cisternae of the endoplasmic reticulum. Particles were observed budding both from limiting cell membranes and from intracellular vesicles of tumour cells. FINKEL and BISKIS (1969) described the particles, found in 90Sr bone tumours of mice, as resembling FBJ virus. This virus which has been isolated from a spontaneous osteosarcoma of a CF 1 mouse (FINKEL et aI., 1966) is, morphologically, a type C virus producing bone tumours in mice (BrsKls and FINKEL, 1969; YUMOTO et ai., 1970). Concerning the occurrence of FBJ virus-like particles in murine 90Sr bone tumours, FINKEL and BISKIS (1969) suggested that 90Sr usually might induce osteogenic sarcomas by inactivating a viral inhibitor. The particles, found in 224Ra osteosarcomas and a metastatic tumour of the liver, also differ in their morphology and formation from those virus-like particles which have recently been revealed in cells from two osteosarcomas induced in CBA mice by 226Ra and 90Sr (LLOYD and MACKEVICIUS, 1971). These intracytoplasmic "vesicular bodies" or "coated vesicles", also found in FBJ virus-induced osteosarcomas of CBA mice, were about 100 m!! in diameter and some appeared to bud from the cell membrane into the cytoplasm. Nothing can be deduced, from the present electron microscopic observations, regarding the significance of the intracellular doughnut-shaped particles found in 224Ra-induced osteosarcomas of three NMRI mice and in a liver metastasis of one of the animals. It is not known at the moment whether the particles resembling type A viral particles are involved in the pathogenesis of the bone tumours.
Virus-Like Particles in Osteosarcomas . 125
The significance of type A particles in murine malignant tumours, especially in relation to oncogenesis, has been discussed by many authors (BERNHARD, 1960; DALTON et aI., 1961; PEARSON and BAKER, 1961; DALTON, 1962; DE HARVEN, 1968; KODAMA and KODAMA, 1973). As type A particles do not possess a central electron-dense nucleoid which presumably contains the viral reproductive nucleic acid, they appear to be probably incomplete and noninfectious viral particles (DALTON et aI., 1961; PEARSON and BAKER, 1961). KODAMA and KODAMA (1973) recently found a significant quantitative correlation between the degree of tumour invasiveness and the content of intracisternal type A viral particles in Ehrlich ascites tumours of mice. The authors discussed the possibility that the type A particle might represent a precursor form of a virus.
Zusammenfassung Die elektronenmikroskopisdte Untersudtung Ra-124-induzierter O steosa rkome dreier Mause und einer Osteosarkc mmetastase in der Leber eines der Tiere ergab, daB in allen Tumoren eigenartige intrazellula re Partikel vorhand en w aren. Die Partikel waren rund , hatten einen Durdtmesser von etwa 80 nm und bestanden aus zwei elektronendichten, ringformigen, membranartigen Sdtalen, die ein helles Zentrum kon ze ntrisdt umgaben. Fast aile di eser "doughnut" -ahnlidt aussehenden Partikel befanden sich in Zisternen des rauhen endoplasmatischen Retikulums (RER) von Tumorzellen. Selten sah man sie in intrazytopla ~ matisdten glattwandigen Vakuolen oder frei in der zytoplasmatischen Matrix lokalisiert. Gelegentlidt wurden halbrunde Pa rtikel gefunden, die sich vermutlich gerade durdt Knospung in Zisternen des RER hinein von dessen Membrankomponente bildeten. Die Partikel haben auf Grund ihrer Ultrastruktur, GroBe und Entstehungsart sowie ihrer lokalisaticn z u intrazellula ren Strukturen Ahnlidtkeit mit A-Typ-Viruspa rtikeln . Die Bedeutung der virusahnlidten P artikel, vor allem im Hinblick auf die Pathogenese der malignen Knodtentumoren , ist gegenwartig nidtt bekannt. Acknowledgements The author expresses sincere th a nks to Miss W. SCHAEDEL for skillful technical assistance a nd to Dr. S. E. NICHOLLS for revision of the manuscript.
References BERNHARD, W. : Cancer R es. 18,491 (1958) BERNHARD, W.: Cancer Res. 20, 712 (1960) BERNHARD, W. and GUERIN, M.: C. R. Acad. Sci. (Paris) 247, 1802 (1958) BERNHARD, W. and GROSS, L. : C. R. Acad. Sci. (Paris) 248, 160 (1959) BISKIS, B. O. and FINKEL, M. P. : In "Proc. of the 27th Annual Meeting of the Electron Microscopy Soc. of America" (ARCENEAUX, c.]., ed.), pp. 384-385 . Claitor's Publishing Co., Baton Rouge (1969) DALTON, A. ].: Fed. Proc. 21 , 936 (1962)
I26 . K.-H. MARQUART DALTON, A. J.: J. nat. Cancer Inst. 49, 323 (1972) DALTON, A. J., POTTER, M., and MERWIN, R. M.: J. nat. Cancer Inst. 26, 1221 (1961) DE HARVEN, E.: In "Experimental Leukemia" (RICH, M. A., ed.), pp. 97-129. NorthHollan d Publishing Co., Amsterdam (1968) FINKEL, M. P. and BISKIS, B. 0.: Acta Un. into Cancr. 15, 99 (1959) FINKEL, M. P. and BISKIS, B. 0.: Progr. expo Tumor Res. 10, 72 (1968) FINKEL, M. P. and BrsKls, B. 0.: In "Delayed Effects of Bone-Seeking Radionuclides " (MAYS, C. W., JEE, W. S. S., LLOYD, R. D., STOVER, B. J., DOUGHERTY, J. H., and TAYLOR, G. N., eds.), pp. 417-435. University of Utah Press, Salt Lake City (1969) FINKEL, M. P., BISKIS, B. 0., and JINKINS, P. B.: Science I5I, 698 (1966) FINKEL, M. P., BISKIS, B. 0 ., and JINKINS, P. B.: In "Radiation-Induced Cancer" (Internat. Atomic Energy Agency, ed.), pp. 369-385. Vienna (1969) GAUGAS, J. M., CHESTERMAN, F. c., HIRSCH, M. S., REES, R. J. W., HARVEY, J. J., and GILCHRIST, c.: Nature 221,1033 (1969) GOSSNER, W., HINDRINGER, B., HUG, 0 ., Luz, A., and MULLER, W. A.: In "4th Internat. Congo of Radiation Research", book of abstracts, p. 84. Evian (1970) GOSSNER, W., HINDRINGER, B., HUG, 0., Luz, A., and MULLER, W. A.: In "Contamination by Bone-Seeking Radionuclides and Radioprotection" (Soc. Franc. de Radioprotection, ed.), pp. 528-548. Paris (1971 a) GassNER, W., HINDRINGER, B., HUG, 0., Luz, A., and MULLER, W. A.: In "8th Annual Meeting of the Europ. Soc. for Radiation Biology", book of abstracts, p. 50. Basko Polje (1971 b) GOSSNER, W., HINDRINGER, B., Luz, A., and SCHWABE, M.: Z. Krebsforsch. 78, 225 (1972) HUG, 0., GessNER, W., MULLER, W. A., Luz, A., and HIND RINGER, B.: "RadiationInduced Cancer" (Internat. Atomic Energy Agency, ed.), pp. 393-409. Vienna (1969) KODAMA, T. and KODAMA, M.: J. nat. Cancer Inst. 50, 707 (1973) LLOYD, E. and MACKEVICIUS, F.: In "Argonne National Laboratory, Radiological Physics Division, Annual Report" (Argonne National Laboratory, ed.), ANL 7860, Part II, pp. 206- 23 I. Argonne (1971) Luz, A., MULLER, W. A., GOSSNER, W., HINDRINGER, B., and HUG, 0.: In "9th Annual Meeting of the Europ. Soc. for Radiation Biology", book of abstracts, p. 103. Rome (197 2) MULLER, W. A.: Int. J. Radiat. Bio!. 20, 27 (1971) NILSSON, A.: Acta radio!. 9,155 (1970) PEARSON, H. E. and BAKER, R. F.: J. nat. Cancer Inst. 27, 793 (1961) SIMPSON, E. and NEHLSEN, S. L.: Clin. expo Immuno!. 9, 79 (1971) SJOGREN, H. O. and RINGERTZ, N.: J. nat. Cancer Inst. 28,859 (1962) STEWART, S. E., EDDY, B. E., and BORGESE, N.: J. nat. Cancer Inst. 20,1223 (1958) VAN PUTTEN, L. M. and DE VRIES, M. J.: J. nat. Cancer Inst. 28,587 (1962) YUMOTO, T., POEL, W. E., KODAMA, T., and DMOCHOWSKI, L.: Tex. Rep. Bio!. Med. 28,145 (1970) Dr. K.-H. MARQUART, Department of General and Experimental Pathology, Institute of Biology, Gesellschaft fur Strahlen- und Umweltforschung mbH Munchen, D-8042 Neuherberg, Ingolstadter Landstra~e I, Germany
Department of General and Experimental Pathology (Head of Dept.: Prof. Dr. W. GassInstitute of Biology, Gesellschaft fur Strahlen- und Umweltforschung mbH Munchen
NER),
Virus-Like Particles in O steosarcomas * **
224
Ra-Induced Murine
Virusahnliche Partikel in Ra-224-induzierten Osteosarkomen der Maus K.-H. MARQUART With 5 Figures' Received November
I,
1973 . Accepted February
II,
1974
Summary Electron microscopic investigation of osteosarcomas from three mice, induced by the incorporation of 224Ra, and an osteosarcoma metastasis from the liver of one of the animals revealed the presence of peculiar intracellular particles in all of the tumours. The particles were spherical, measured about 80 nm in diameter and consisted of two electron-dense, ring-shaped, membrane-like shells which concentrically surrounded an electron-lucent core. Nearly all of the particles with this doughnut-like ultrastructural appearance occurred in cisternae of the rough-surfaced endoplasmic reticulum (RER) of tumour cells. They were seldom observed in intracytoplasmic smooth-surfaced vacuoles or located freely in the cytoplasmic matrix. Crescent-shaped particles were occasionally found presumably in the stage of formation by budding into cisternae of the RER from its membrane component. The ultrastructure, size and formation of the particles, as well as their location in relation to intracellular structures, are similar to those described for type A viral particles. The significance of the virus-like particles, especially in relation to the pathogenesis of the malignant bene tumours, is not known at present.
Malignant bone tumours arise in mice as late effects following the incorporation of bone-seeking radionuclides such as 905r, 226Ra, 224Ra or 227Th (FINKEL and BISKIS, 1959, 1968, 1969; VAN PUTTEN and DE VRIES, 1962; FINKEL * Dedicated to Prof. Dr. O. HUG on the occasion of his 60th birthday. ~ ..
Performed within the framework of an association contract EURA TOM-GSF (Con-
tract No. 090-72-1 BIAD).
Virus-Like Particles in Osteosarcomas . 117
et aI., 1969; HUG et aI., 1969; NILSSON, 1970; GaSSNER et aI., 1970, 1971 a, b). It is still not known whether the radiation, emitted from the incorporated radionuclides, induces osteosarcomas directly by possible biochemical lesions or indirectly by suppressing the immune response and unmasking a latent oncogenic virus infection. Since experiments have shown that malignant bone tumours can be induced in mice by the inoculation of viruses (STEWART et aI., 1958; SJOGREN and RINGERTZ, 1962; FINKEL et aI., 1966; YUMOTO et aI., 1970) or by immunosuppression (GAUGAS et aI., 1969; SIMPSON and NEHLS EN, 1971), the hypothesis of the viral origin of radiation-induced murine osteosarcomas has received greater attention. FINKEL and BISKIS (1969) have seen, in ultrathin sections of 90Sr bone tumours from mic~, particles which were similar in appearance to viral particles found in virus-induced murine bone tumours. Recently, membraneous structures resembling viral particles have been observed in primary or passaged osteosarcomas of mice, induced by the incorporation of 226Ra or 90Sr as well as by the inoculation of an oncogenic RNA virus (LLOYD and MACKEVICIUS, 1971). The present study describes the occurrence of virus-like particles in osteosarcomas from three mice, induced by the incorporation of 224Ra, and a metastatic tumour from the liver of one of the animals.
Materials and Methods The three mice belonged to a dose-protraction experiment carried out with the bone tumour-inducing 224Ra (Luz et aI., 1972). The ultrastructure of 224Ra-induced murine osteosarcomas was studied within the framework of this experiment. The present study is a preliminary report describing a finding which appeared to be of sufficient interest to be presented at this time. Four-week-old, female NMRI mice (Neuherberg strain) were injected, intraperitoneally, with 224Ra. 224Ra is a short-lived (half-life 3.64 days), alpha-emitting radionuclide. It is a bone volume-seeker which mainly deposits after injection into mice in sites similar to those in which calcium is deposited. Two mice of the present study received 12 ftCi/kg of body weight 224Ra protracted over a period of 4 weeks (the individual amounts of activity were 1.5 ftCi/kg each 3.5 days). One mouse was injected with 36 ftCi/kg of body weight 224Ra also protracted over 4 weeks (the individual amounts of activity were 4.5 ftCi/kg each 3.5 days). The activity of 12 ftCi/kg 224Ra, injected into a mouse, produces after total decay an accumulated average skeletal dose of about 360 rads, the activity of 36 ftCi/kg 224Ra one of about 1080 rads (MULLER,
197 1). 224Ra (Thorium X) was used as commercially supplied (Amersham Buchler GmbH & Co. KG, Braunschweig, Germany) in the form of a radium chloride solution containing 2-6.2 mg/ml calcium chloride. The solution contained impurities consisting of the long-lived radionuclides 228Th, 226Ra and 210Pb whose total activities were not more than 5 X 10-6 Ci per Ci 224Ra (MULLER, 1971). Before administration to the animals, the radium chloride solution was diluted with physiological saline to obtain the different amounts of 224Ra.
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8 . K.-H.
MARQUART
The three injected mice were maintained on a standard pellet diet together with drinking water ad libitum. Eighteen months following the first 224Ra injection a protruberant tumour had developed in the left pelvic region of one animal which had been injected with 12 !lei/kg 224Ra. Autopsy of the mouse, after sacrifice using chloroform, and light microscopic studies revealed an osteoblastic osteosarcoma of the proximal part of the left femur and metastatic tumours in the liver. In the other mouse which had received 12 !lCi/kg 224Ra a tumour was recognizable in the medial pelvic region 19 months following the first incorporation of 224Ra. Autopsy of the animal showed an osteoblastic osteosarcoma of the os sacrum. Twenty months after the first 224Ra injection a prot rube rant tumor was found in the right lower head region of the animal which had been injected with 36 !lCi/kg 224Ra. Autopsy of the mouse revealed a fibroblastic osteosarcoma of the mandibula. This type of osteosarcoma is typical for 224Ra-induced murine osteosarcomas of the head region which scarcely show osteoblastic differentiation as do bone tumours of other skeletal sites (GassNER et a!., 1972). Neither leukemia nor any other pathologic alterations were found in the three tumour-bearing mice. Immediately after sacrifice of the mice, small pieces of tumour tissues, from the three osteosarcomas and one liver metastasis, were removed and placed in cold phosphate-buffered 6.25% glutaraldehyde (pH 7.3) or in the case of the tissue from the fibroblastic osteosarcoma of the mandibula in cold phosphate-buffered 1% osmium tetroxide (pH 7.3). Immersed in the fixative solutions the specimens were cut into cubes of about I mm 3. The tissue cubes were fixed in the glutaraldehyde for 4 hours followed by postfixation in cold phosphate-buffered 10f0 osmium tetroxide for 2 hours. The tissue cubes from the tumour of the lower jaw were primarily fixed in the osmium tetroxide for 2 hours. Following dehydration through a graded series of alcohols to propylene oxide, the specimens were embedded in Epon 8 I2. Sections, cut with a diamond knife on a Reichert Om U2 microtome, were stained with uranyl acetate and lead citrate and examined in an AEI EM 6B electron microscope.
Results Electron microscopic investigation of the tumour tissues from the three 224Ra-induced murine osteosarcomas and the liver metastasis revealed the presence of peculiar intracellular particles which were only visible at a magnification higher than I : 15,000. The occurrence of these structures in the different tumours varied slightly. Osteoblastic osteosarcoma of the femur. Many of the osteoblast-like cells from the osteosarcoma of the femur contained electron-dense, spherical particles measuring about 80 nm in diameter (Fig. I). The particles usually occurred singly (Fig. I, 2), but in a few cells groups of them were present. Higher magnification revealed that the particles consisted of two electron-dense, ring-shaped, membrane-like shells which concentrically surrounded a relatively electron-lucent core measuring approximately 45 nm in diameter (Fig. 2, 3). The inner membrane-like shell bounded the electron-lucent core and the outer one limited the particles. Occasionally, the inner shell
Virus-Like Particles in Osteosarcomas .
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Fig. r. A low magnification electron micrograph showing portions of two osteoblast-like cells from a 224Ra-induced murine osteosarcoma of the femur. In one cell a single electrondense, spherical particle (arrow) can be detected in a cisterna of the rough-surfaced endoplasmic reticulum. B, mineralized bone matrix. X 16,500.
appeared very electron-dense whereas the outer one was less clearly defined (Fig. 3). Nearly all of the particles with this so-called "doughnut-like" ultrastructural appearance were present in cisternae of the rough-surfaced endoplasmic reticulum (RER) of osteosarcoma cells (Fig. I-3). They were seldom observed free in the cytoplasmic matrix or in intracytoplasmic membranebound vacuoles (Fig. 3). In very few cells, the doughnut-like particles could also be detected within the perinuclear space. None of the particles were observed in intercellular spaces of the osteosarcoma tissue.
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Fig. 2. Two doughnut-shaped particles (arrows) in cisternae of the RER of an osteosarcoma cell at higher magnification . Their electron-lucent core is clearly visible. X 60,000.
Crescent-shaped particles were occasionally found presumably in the stage of formation by budding into cisternae of the RER from its membrane component (Fig. 3). Various stages in this budding process were seen. Particles developed only from membranes of the RER. Other cellular membranes such as those limiting cytoplasmic vacuoles or the limiting cell membrane were never involved in this process. Osteosarcoma metastasis 0/ the liver. Doughnut-like particles, identical to those from the osteosarcoma of the femur, were also found in osteoblast-like cells from the metastatic tumour in the liver of the same animal. The particles were seen only rarely in cells from this tumour, and then in cisternae of the RER and in a few cases within the cytoplasmic matrix. No particles were observed in intracytoplasmic vacuoles and none were found budding into cisternae of the RER. Doughnut-shaped particles were never detected in intercellular spaces of the tissue from the liver metastasis. In cells of the liver tissue, adjacent to the metastatic tumour, no such particles were observed.
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Fig. 3. A doughnut-like particle (arrow) can be observed in an intracytoplasmic mcmbranebound vacuole. Another particle (arrowhead) is located in a cisterna of the RER. A third particle (double arrow) is found budding into the RER from its membrane component. d, dense body. X 8o,oco.
Osteoblastic osteosarcoma of the os sacrum. Electron microscopic investigation of tissue from the osteosarcoma of the os sacrum also revealed the presence of intracellular doughnut-shaped particles identical in their ultrastructure to those described above. However in this tumour, relatively few of the particles were present. They occurred only singly in cisternae of the RER (Fig. 4) and seldom freely in the cytoplasm of a few osteoblast-like cells. No budding particles were seen and none of the particles were found in extracellular spaces of the tissue. Fibroblastic osteosarcoma of the mandibula. Doughnut-shaped particles, identical in structure and size to those which occurred in the tumours mentioned above, were detected in many of the cells from the osteosarcoma
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Fig. 4. A part of an osteoblast-like cell from a 224Ra-induced murine osteosarcoma of the os sacrum is shown. A doughnut-shaped particle (arrow) can be observed in a cisterna of the RER. Trabecular mineralized bone matrix (B) is closely apposed to the cellular border. X 60,000.
of the mandibula. The particles were as frequent in this tumour as in the osteoblastic osteosarcoma of the femur. They occurred mainly in cisternae of the RER (Fig. 5) of tumour cells. Very few particles were located freely in the cytoplasmic matrix and a single particle was found in a smoothsurfaced vacuole of a Golgi complex. Crescent-shaped particles in process of budding into cisternae of the RER from its membrane component were frequently seen (Fig. 5). Ribosomes were absent from the membranes of the RER on sites of particle formation. Occasionally, the cisternal membranes from which particles developed were abnormally thickened (Fig. 5). None of the particles occurred in extracellular spaces of the tumour tissue. The average outer diameter of all particles, found in the three 224Ra_ induced murine osteosarcomas and the liver metastasis, was 80 nm, ranging from 70 to 90 nm. The average diameter of the electron-lucent core of the particles was 45 nm, ranging from 40 to 55 nm.
Virus-Like Particles in Osteosarcomas .
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Fig. 5. Several doughnut-like particles are located in cisternae of the RER of a cell from a 224Ra-induced murine ostecsarcoma of the mandibula. One particle (arrow) appears to bud from a membraneous stalk into a cisterna of the RER. The membrane of this cisterna is abnormally thickened and lacks typically aligned ribosomes. X 80,000.
Discussion The particles, found in ultrathin sections of three 224Ra-induced murine osteosarcomas and a liver metastasis, show the following morphological characteristics: 1. The particles occur only intracellularly. 2. They are spherical and consist of two electron-dense, ring-shaped, membrane-like shells which concentrically surround an electron-lucent core (doughnut-like ultrastructural appearance). 3. The average diameter of the particles is 80 nm, that of the electron-lucent core 45 nm. 4. Nearly all of the particles occur in cisternae of the RER of tumour cells. They are seldom observed in smoothsurfaced vacuoles or located freely in the cytoplasmic matrix. 5. Crescentshaped particles are occasionally found presumably in the stage of formation by budding into cisternae of the RER from its membrane component. The ultrastructure, size and formation of the particles, as well as their location in relation to intracellular structures, are similar to those described for type A viral particles (BERNHARD, 1958; BERNHARD and GUERIN, 1958;
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DALTON, 1962, 1972). Following the sub-classification of DALTON (1962) the particles resemble type As viral particles. The occurrence of type A particles in radiation-induced osteogenic sarcomas has not yet been reported. Such viral particles have been described in cells of various other mouse neoplasms such as mammary carcinomas, Ehrlich ascites tumours, melanomas, leukemias, plasma-cell tumours, salivary gland tumours, precancerous hyperplastic mammary tissues, lymphomas and mastocytomas (for references see BERNHARD, 1958, 1960; BERNHARD and GROSS, 1959; DALTON et aI., 1961; PEARSON and BAKER, 1961; DALTON, 1962; DE HARVEN, 1968). The virus-like particles which have been revealed by FINKEL and BrSKrs (1969) in osteosarcomas induced in CFI, CBA and X/Gf mice by the incorporation of 90Sr are different in their ultrastructural appearance from those reported in the present study. In contrast to the observed occurrence of viruslike particles only within cells of 224Ra bone tumours, these authors saw doughnut-like particles, consisting of three concentric membrane-like shells, in extracellular spaces as well as in cisternae of the endoplasmic reticulum. Particles were observed budding both from limiting cell membranes and from intracellular vesicles of tumour cells. FINKEL and BISKIS (1969) described the particles, found in 90Sr bone tumours of mice, as resembling FBJ virus. This virus which has been isolated from a spontaneous osteosarcoma of a CF 1 mouse (FINKEL et aI., 1966) is, morphologically, a type C virus producing bone tumours in mice (BrsKls and FINKEL, 1969; YUMOTO et ai., 1970). Concerning the occurrence of FBJ virus-like particles in murine 90Sr bone tumours, FINKEL and BISKIS (1969) suggested that 90Sr usually might induce osteogenic sarcomas by inactivating a viral inhibitor. The particles, found in 224Ra osteosarcomas and a metastatic tumour of the liver, also differ in their morphology and formation from those virus-like particles which have recently been revealed in cells from two osteosarcomas induced in CBA mice by 226Ra and 90Sr (LLOYD and MACKEVICIUS, 1971). These intracytoplasmic "vesicular bodies" or "coated vesicles", also found in FBJ virus-induced osteosarcomas of CBA mice, were about 100 m!! in diameter and some appeared to bud from the cell membrane into the cytoplasm. Nothing can be deduced, from the present electron microscopic observations, regarding the significance of the intracellular doughnut-shaped particles found in 224Ra-induced osteosarcomas of three NMRI mice and in a liver metastasis of one of the animals. It is not known at the moment whether the particles resembling type A viral particles are involved in the pathogenesis of the bone tumours.
Virus-Like Particles in Osteosarcomas . 125
The significance of type A particles in murine malignant tumours, especially in relation to oncogenesis, has been discussed by many authors (BERNHARD, 1960; DALTON et aI., 1961; PEARSON and BAKER, 1961; DALTON, 1962; DE HARVEN, 1968; KODAMA and KODAMA, 1973). As type A particles do not possess a central electron-dense nucleoid which presumably contains the viral reproductive nucleic acid, they appear to be probably incomplete and noninfectious viral particles (DALTON et aI., 1961; PEARSON and BAKER, 1961). KODAMA and KODAMA (1973) recently found a significant quantitative correlation between the degree of tumour invasiveness and the content of intracisternal type A viral particles in Ehrlich ascites tumours of mice. The authors discussed the possibility that the type A particle might represent a precursor form of a virus.
Zusammenfassung Die elektronenmikroskopisdte Untersudtung Ra-124-induzierter O steosa rkome dreier Mause und einer Osteosarkc mmetastase in der Leber eines der Tiere ergab, daB in allen Tumoren eigenartige intrazellula re Partikel vorhand en w aren. Die Partikel waren rund , hatten einen Durdtmesser von etwa 80 nm und bestanden aus zwei elektronendichten, ringformigen, membranartigen Sdtalen, die ein helles Zentrum kon ze ntrisdt umgaben. Fast aile di eser "doughnut" -ahnlidt aussehenden Partikel befanden sich in Zisternen des rauhen endoplasmatischen Retikulums (RER) von Tumorzellen. Selten sah man sie in intrazytopla ~ matisdten glattwandigen Vakuolen oder frei in der zytoplasmatischen Matrix lokalisiert. Gelegentlidt wurden halbrunde Pa rtikel gefunden, die sich vermutlich gerade durdt Knospung in Zisternen des RER hinein von dessen Membrankomponente bildeten. Die Partikel haben auf Grund ihrer Ultrastruktur, GroBe und Entstehungsart sowie ihrer lokalisaticn z u intrazellula ren Strukturen Ahnlidtkeit mit A-Typ-Viruspa rtikeln . Die Bedeutung der virusahnlidten P artikel, vor allem im Hinblick auf die Pathogenese der malignen Knodtentumoren , ist gegenwartig nidtt bekannt. Acknowledgements The author expresses sincere th a nks to Miss W. SCHAEDEL for skillful technical assistance a nd to Dr. S. E. NICHOLLS for revision of the manuscript.
References BERNHARD, W. : Cancer R es. 18,491 (1958) BERNHARD, W.: Cancer Res. 20, 712 (1960) BERNHARD, W. and GUERIN, M.: C. R. Acad. Sci. (Paris) 247, 1802 (1958) BERNHARD, W. and GROSS, L. : C. R. Acad. Sci. (Paris) 248, 160 (1959) BISKIS, B. O. and FINKEL, M. P. : In "Proc. of the 27th Annual Meeting of the Electron Microscopy Soc. of America" (ARCENEAUX, c.]., ed.), pp. 384-385 . Claitor's Publishing Co., Baton Rouge (1969) DALTON, A. ].: Fed. Proc. 21 , 936 (1962)
I26 . K.-H. MARQUART DALTON, A. J.: J. nat. Cancer Inst. 49, 323 (1972) DALTON, A. J., POTTER, M., and MERWIN, R. M.: J. nat. Cancer Inst. 26, 1221 (1961) DE HARVEN, E.: In "Experimental Leukemia" (RICH, M. A., ed.), pp. 97-129. NorthHollan d Publishing Co., Amsterdam (1968) FINKEL, M. P. and BISKIS, B. 0.: Acta Un. into Cancr. 15, 99 (1959) FINKEL, M. P. and BISKIS, B. 0.: Progr. expo Tumor Res. 10, 72 (1968) FINKEL, M. P. and BrsKls, B. 0.: In "Delayed Effects of Bone-Seeking Radionuclides " (MAYS, C. W., JEE, W. S. S., LLOYD, R. D., STOVER, B. J., DOUGHERTY, J. H., and TAYLOR, G. N., eds.), pp. 417-435. University of Utah Press, Salt Lake City (1969) FINKEL, M. P., BISKIS, B. 0., and JINKINS, P. B.: Science I5I, 698 (1966) FINKEL, M. P., BISKIS, B. 0 ., and JINKINS, P. B.: In "Radiation-Induced Cancer" (Internat. Atomic Energy Agency, ed.), pp. 369-385. Vienna (1969) GAUGAS, J. M., CHESTERMAN, F. c., HIRSCH, M. S., REES, R. J. W., HARVEY, J. J., and GILCHRIST, c.: Nature 221,1033 (1969) GOSSNER, W., HINDRINGER, B., HUG, 0 ., Luz, A., and MULLER, W. A.: In "4th Internat. Congo of Radiation Research", book of abstracts, p. 84. Evian (1970) GOSSNER, W., HINDRINGER, B., HUG, 0., Luz, A., and MULLER, W. A.: In "Contamination by Bone-Seeking Radionuclides and Radioprotection" (Soc. Franc. de Radioprotection, ed.), pp. 528-548. Paris (1971 a) GassNER, W., HINDRINGER, B., HUG, 0., Luz, A., and MULLER, W. A.: In "8th Annual Meeting of the Europ. Soc. for Radiation Biology", book of abstracts, p. 50. Basko Polje (1971 b) GOSSNER, W., HINDRINGER, B., Luz, A., and SCHWABE, M.: Z. Krebsforsch. 78, 225 (1972) HUG, 0., GessNER, W., MULLER, W. A., Luz, A., and HIND RINGER, B.: "RadiationInduced Cancer" (Internat. Atomic Energy Agency, ed.), pp. 393-409. Vienna (1969) KODAMA, T. and KODAMA, M.: J. nat. Cancer Inst. 50, 707 (1973) LLOYD, E. and MACKEVICIUS, F.: In "Argonne National Laboratory, Radiological Physics Division, Annual Report" (Argonne National Laboratory, ed.), ANL 7860, Part II, pp. 206- 23 I. Argonne (1971) Luz, A., MULLER, W. A., GOSSNER, W., HINDRINGER, B., and HUG, 0.: In "9th Annual Meeting of the Europ. Soc. for Radiation Biology", book of abstracts, p. 103. Rome (197 2) MULLER, W. A.: Int. J. Radiat. Bio!. 20, 27 (1971) NILSSON, A.: Acta radio!. 9,155 (1970) PEARSON, H. E. and BAKER, R. F.: J. nat. Cancer Inst. 27, 793 (1961) SIMPSON, E. and NEHLSEN, S. L.: Clin. expo Immuno!. 9, 79 (1971) SJOGREN, H. O. and RINGERTZ, N.: J. nat. Cancer Inst. 28,859 (1962) STEWART, S. E., EDDY, B. E., and BORGESE, N.: J. nat. Cancer Inst. 20,1223 (1958) VAN PUTTEN, L. M. and DE VRIES, M. J.: J. nat. Cancer Inst. 28,587 (1962) YUMOTO, T., POEL, W. E., KODAMA, T., and DMOCHOWSKI, L.: Tex. Rep. Bio!. Med. 28,145 (1970) Dr. K.-H. MARQUART, Department of General and Experimental Pathology, Institute of Biology, Gesellschaft fur Strahlen- und Umweltforschung mbH Munchen, D-8042 Neuherberg, Ingolstadter Landstra~e I, Germany