[CANCER RESEARCH 35, 2000-2006, August 1975]

SUMMARY

Nuclei of human neurilemoma cells exhibit deep andextensive invaginations of part of their surface. Suchinvaginations contain cytoplasmic matter. However, inareas of the nucleoplasm distant from the invaginations,small membrane-bound bodies, some of which contain a“nucleoid,―occur either singly or grouped together andenclosed within a large membrane body. These small bodiesare not considered virus-like. Degenerated nuclei fromcultured tumor ti .sue contain spherical bodies, 130 to 230nm in diamter, with spikes on their surface similar to thoseseen on envelopes of herpes-type viruses. Significance ofthese bodies in vivo and in vitro tumor tissues is not known.

INTRODUCTION

Variations in ultrastructural nuclear morphology areknown to occur in pathological conditions. Most commonamong these are certain inclusions generally referred to asnuclear bodies and alterations in the nuclear surface configuration. Whereas many different forms of nuclear bodieshave been described in various neoplastic diseases of man(4—6,16, 23, 25, 26), indentations and projections of nuclearenvelope are mostly found in lymphoproliferative neoplasms (1, 12, 18), nasopharyngeal carcinoma (14), and incells infected with herpes-type virus (8, 9, 13, 22). This studydescribes both these ultrastructural characteristics in humanneurilemoma cell nuclei and some unusual spherical structures in degenerated nuclei in cultured tumor tissue.

MATERIALS AND METHODS

Tumor Tissue. Samples of neurilemoma tissue from 3patients were received from surgery in sterile 0.01 M phosphate-buffered saline containing penicillin and streptomycin. The tumor was very soft and a piece of it wasprocessed for electron microscopy while the rest was usedfor in vitro cultivation. Growth and morphological characteristics of cultured neurilemoma cells will be describedelsewhere (manuscript in preparation).

Electron Microscopy. Tumor tissue was fixed overnight in2.5% glutaraldehyde in phosphate-buffered saline. It wassliced into approximately I-cu mm pieces and washed twice

1This investigation was supported in part by the American CancerSociety, Illinois Division, and the Teaching and Research Fund of MercyHospital and Medical Center.

Received August 29, 1974; accepted April 28, 1975.

in phosphate-buffered saline. It was postfixed for 1 hr inchromeosmium and after dehydration in graded concentrations of alcohol it was embedded in Epon-Aralditemixture. Sections 1 to 1.5 sm thick were cut on glass knives,stained with toluidine blue (7), and examined under alight microscope. Thin sections were cut on an LKBUltrotome with a diamond knife, stained with lead anduranyl acetate, and examined under a Siemens-ElmiskopI electron microscope at an accelerating voltage of 80 kV.

RESULTS

Neurilemoma cells have oval nuclei in which the chromatin material is slightly marginated and also distributedthroughout the nucleoplasms in small clumps (Figs. 1 to 4).The nucleolus is large and has the characteristic granularappearance. The cells were characterized as tumor cellsbecause of the presence of (a) basement membrane aroundmost of them, a fact which has supported the hypothesisthat neurilemoma is derived from Schwann cells, and (b)specific granules not present in fibroblast cells of thestroma.

Nuclear Invaginations. Tumor cell nuclei generally havesmooth surfaces; however, in some areas the nuclear surfaceis invaginated. Nuclear invaginations vary both in depth andin number (Figs. 1 to 6). Approximately 10% of nuclei inany field exhibit such invaginations. Since a part of anucleus appears to be indented (Figs. 1 and 2) only certainplanes of sectioning will contain indented nuclei, while inother planes the same nucleus will appear round or oval; amuch greater percentage of tumor cell nuclei could therefore be invaginated.

Nuclear invaginations have bizarre shapes (Figs. 2 and 3)within the nucleoplasm, suggesting either a number of deepinvaginations over a certain area of nuclear surface orextensive branching of an invagination within the bulk of anucleus. Since the invaginations are lined with nuclearmembrane with attached chromatin material, cytoplasmicmatter can sometimes be seen within such invaginations(Figs. 2 to 5, Cy). In some areas, on the other hand, thedouble nuclear membrane with its attached chromatin is notseen and it becomes difficult to attribute precisely thepartially enclosed matter as cytoplasmic (Figs. 3 and 4,arrows). Curved arrows (Fig. 3) point to material thatappears similar to that within nuclear invaginations in otherareas but is not enclosed by a nuclear envelope. The electronmicrographs (Figs. 2 to 5) also exhibit the presence of amore conspicuous and abundant amount of perichromatinmaterial along the nuclear invagination membranes than

2000 CANCER RESEARCH VOL. 35

Ultrastructural Characteristics of Human Neurilemoma Cell Nuclei'

Satish Chandra, Michael J. Jerva, and Jack D. Clemis

Departments ofResearch [S. C.] and Surgery [M. J. J., J. D. C.], Mercy Hospital and Medical Center, Chicago, Illinois 60616

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Human Neurilemoma Cell Nuclei

along the non-nuclear invagination membranes of tumorcells.

Dense osmiophilic bodies are sometimes seen within thenuclear invaginations. Some of these are membrane bound,while others are irregular, very osmiophilic, and not distinctly membrane bound (Figs. 3 and 4, heavy arrows).Some lie in nucleoplasm with only a single membranearound them, (Fig. 4, arrows).

Projection of Nuclear Envelope. Because of extensiveinvaginations of neurilemoma nuclei, projections of nuclearenvelope are often observed (Figs. I , 3, 5, and 6, NP).Occasionally, these projections are large and connect 2lobes of a nucleus (Fig. I, right).

Small Membrane-bound Bodies. Nuclei of neurilemomacells also contain small membrane-bound bodies approximately 130 nm in size. Some of these contain a dense“nucleoid.―They occur in clusters either free in thenucleoplasm (Figs. S and 6, arrows) or enclosed within alarge membrane-bound body (Figs. 5 and 6, heavy arrows).Such bodies do not occur within nuclear invaginations andwere not observed in the cytoplasm of tumor cells.

Nuclear Bodies. Nuclear bodies similar to those describedby other investigators (5, 16) are found in nuclei ofneurilemoma cells. These are nearly spherical bodies of finefibrous material that gives them a whorl-like appearance.They often contain small spherical bodies (Figs. 7 and 8)some of which may be bound by a membrane. In Fig. 7small dense bodies (arrows) measuring about 45 nm have anelectron-lucent substance around them and are embedded inan amorphous matter that does not appear to be chromatin.The latter has a granular matrix (Fig. 7, heavy arrow).Occasionally, nuclear bodies also contain segments ofmembrane (Fig. 7, M). Similar membrane segments arealso found free in the nucleoplasm. Fig. 8 shows a membrane-bound spherical body, measuring approximately 120nm, within a nuclear body.

Cultured Cell. Nuclei of cultured neurilemoma cellsgenerally exhibit normal ultrastructural morphology. However, in 5 to 10% of cultured cells the nucleus showsmargination and condensation of chromatin (Fig. 9). Suchcells also contain tubuloreticular structuring or undulatingtubules (9) associated with endoplasmic reticulum in thecytoplasm (Figs. 9 and 10, arrows). In 1 culture, certainareas that represented degenerated nuclei contained unusualspherical bodies of various sizes (Fig. 11). Whereas amajority of these were I 30 to 230 nm in diameter, few largeroval bodies were occasionally seen within the degeneratednuclear mass. These bodies were devoid of any osmiophilicsubstance, and at higher magnifications their surface had abilamellar structure with spikes on the outermost and theinnermost surfaces (Figs. 12 and 13). Such bodies were notseen in healthy cells. Fig. 14 shows for comparison spikes(arrows) on the envelopes of HSV2 type I and on membranes that may be involved in the maturation of HSV type1. Other membranes (heavy arrows) do not contain suchspikes.

2The abbreviation used is: HSV, herpes simplex virus.

DISCUSSION

The most common morphological characteristic of atumor cell nuclei is the increased size of its nucleolus. In theelectron microscope, nuclear bodies have been describedwith increased frequency in pathological and neoplastictissues as compared to normal tissues (5, 16). The formercategory includes viral infection.

Indentation of nuclear membrane and formation oflobules have been described in certain human neoplasms. Inlymphoproliferative cancers, these ultrastructural variationsare consistently observed both in vivo (1 , 12, 14) and in vitro(8, 9, 13, 22). Such variations in nuclear surface were notreported in tumors of the nervous system (21, 25, 26)including a study of 50 cases of human neurilemoma (10,11). It is possible that they escaped detection because oftheir localization on only a part of the nuclear surface asdescribed in the present study.

Nuclear bodies have been observed in diseases associatedwith the human central nervous system, for example, inencephalitis (5) and subacute sclerosing leukoencephalitis(16). Their presence was reported in human brain tumors (5,16, 25, 26) and in experimental lesions produced in brains oflaboratory animals (23). Intranuclear small membranebound spherical bodies (Figs. 5 and 6) which may containsome dense material within them and which may eitheroccur free in the nucleoplasm or within a larger membranebound body have not been described in the human centralnervous system. They are not cytoplasmic bodies since theyare not enclosed by nuclear membrane with the chromatinmaterial on the outside as are areas marked Cy in Figs. 2 to6. The possibility that they represent certain pathologicalchange(s) in an otherwise apparently healthy cell cannot beruled out. Although these membrane-bound bodies havenucleoid and are of the same size as herpes-type viruses,they are not considered virus-like particles.

The significance of nuclear bodies and of small membrane-bound spherical bodies in human neurilemoma tissueis not clear; however, the former has consistently beenobserved in viral infections both in vivo and in vitro,concomitant with the presence of an increased amount ofperichromatin material as in this study. In human epidermaltissue infected with vaccinia virus, nuclear bodies werepresent in the infected cells whereas cells at the periphery ofthe biopsy specimen did not contain these bodies (19).Patrizi and Middelkamp (19) infected cultured WI-26fibroblast cells with vaccinia virus and observed the presence of nuclear bodies in infected cells only. Other virusesthat have been associated with such bodies are adenovirustype 12 (17), simian virus 40 (15), polyoma (3), and herpessimplex and human cytomegaloviruses (20). It is of significance that all these viruses are DNA viruses. It wouldtherefore appear that infection by a DNA virus could beconsidered as a factor in the morphological manifestation ofnuclear bodies in cells. Although no virus has as yet beenimplicated in the genesis of tumors of the human nervoussystem, 2 factors should be considered in this regard: (a)HSV is known to lie dormant in man (2) and (b) HSV cantransform cultured cells to a state of malignancy (24).

AUGUST 1975 2001

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S. Chandraeta!.Furthermore, the presence of tubuloreticular structure incultured cells from the neurilemoma tissues suggests infection by a virus of the herpes group (9) and/or someneoplastic disease process (27). Attempts to isolate aherpes-type virus from cultured neurilemoma cells with cellssusceptible to HSV have so far been unsuccessful. Thesignificance of spherical bodies exhibiting spikes (Figs. 12and 13) in a few degenerated nuclei in neurilemoma cellculture is not known, yet the similarity between these spikesand those on the envelopes of HSV is striking.

Oncogenic viruses generally produce malignant neoplasms. Of the various viruses of the papova group, thepapilloma viruses produce benign lesions in their naturalhosts. On the basis of the present morphological observations, a role, if any, of a DNA virus in the genesis of humanneurilemomas, which are benign in nature, is purely speculative.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical assistance provided byTeresa Valdivieso and Jason Chao.

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2. Bastion, F. 0., Rabson, A. S., Yee, C. L., and Tnalka, T. S.Herpes-virus Hominis: Isolation from Human Tnigeminal Ganglion.Science, 178: 306—307, 1972.

3. Bernhard, W., Fabvre, H. L., and Cramer, R. Electron MicroscopicDemonstration of a Virus in Cells Infected In Vitro with the PolyomaAgent. Compt. Rend., 249: 483—485,1959.

4. Bernhard, W., and Granboulan, N. The Fine Structure of the CancerCell Nucleus. Exptl. Cell Res. Suppl., 9. 19-53, 1963.

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I I. Cravioto, H., and Lockwood, R. The Behavior of Acoustic Neunomain Tissue Culture. Acta Neuropathol., 12: 141—157, 1969.

12. Donfman, R. F. The Fine Structure of a Malignant Lymphoma in aChild from St. Louis, Missouri. J. NatI. Cancer Inst., 38: 491—504,1967.

13. Epstein, M. A., Henle, G., Achong, B. G., and Barr, Y. M.Morphological and Biological Studies on a Virus in Cultured Lymphoblasts from Burkitt's Lymphoma. J. Exptl. Med., 121: 761—770,1965.

14. Gazzolo, L., dc-The, G., Vuillaume, M., and Ho, H. C. Nasopharyngeal Carcinoma. II. Ultrastructure of Normal Mucosa, TumorBiopsies, and Subsequent Epithelial Growth in Vitro. J. NatI. CancerInst., 48: 73—86,1972.

15. Granboulan, N., Tournier, P., Wicker, R., and Bernhard, W. AnElectron Microscope Study of the Development of SV-40 Virus. J.Cell Biol., 17: 423—441,1963.

16. Knishan, A., Uzman, B. G., and Hedley-Whyte, E. T. Nuclear Bodies:

A Component of Cell Nuclei in Hamster Tissues and Human Tumors.J. Ultrastruct. Res., 19: 563—572,1967.

17. LeBuis, J. Alterations in Nuclear and Nucleolar Ultrastructure in KBCells Infected with Adenovirus Type 12. J. Microscop., 5: 59A, 1966.

18. Mollo, F., and Stramignoni, A. Nuclear Projections in Blood andLymph Node Cells of Hman Leukemias and Hodgkin's Disease and inLymphocytes Cultured with Phytohaemagglutin. Brit. J. Cancer, 21:519—523,1967.

19. Patnizi, G., and Middelkamp, J. N. In Vivo and in Vitro Demonstration of Nuclear Bodies in Vaccinia Infected Cells. J. Ultrastruct.Res., 28: 275-287, 1969.

20. Patnizi, G., Middelkamp, J. N., Herweg, J. C., and Thornton, H. K.Human Cytomegalovirus: Electron Microscopy of a Primary ViralIsolate. J. Lab. Clin. Med., 65: 825—838, 1965.

2 1. Pinada, A. Submicroscopic Structure of Acoustic Tumors. Neurology,14: 171—184,1964.

22. Pope, J. H., Achong, B. G., and Epstein, M. A. Cultivation and FineStructure of Virus-Bearing Lymphoblasts from a Second New GuineaBurkitt Lymphoma: Establishment of Sublines with Unusual CultureProperties. Intern. J. Cancer, 3. 171—182, 1968.

23. Popoff, N., and Stewart, S. The Fine Structure of Nuclear Inclusionsin the Brain of Experimental Golden Hamsters. J. Ultrastruct. Res.,23:347—361,1968.

24. Rapp, F., and Duff, R. Transformation of Hamster Embryo Fibroblasts by Herpes Simplex Viruses Type 1 and Type 2. Cancer Res., 33:1527—1534, 1973.

25. Robertson, D. M. Electron Microscopic Studies of Nuclear Inclusionsin Meningiomas. Am. J. Pathol., 45: 835—848, 1964.

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Human Neurilemoma Cell Nuclei

Fig. I . A low-power electron micrograph of human neurilemoma showing 2 tumor cell nuclei with deep indentation (arrow) and projections of nuclearenvelope (NP). x 3,200.

Fig. 2. Extensive cytoplasmic invagination within a tumor cell nucleus which, in this plane, exhibits a smooth surface. Cytoplasmic matter (Cy) withinthe nucleus is demarcated by nuclear envelope with chromatin material attached to it. x 12,000.

Fig. 3. Similar to Fig. 2; however, in certain areas, the cytoplasmic matter (Cy) within the invaginations is not completely enclosed by nuclearmembrane (arrow). Curved arrows, material that appears similar to that within the nuclear invagination in other areas in this field, but is not enclosed bynuclear envelope. Dense osmiophilic bodies are seen within nuclear invaginations (heavy arrows). NP, nuclear envelope projection. x 21,000.

Fig. 4. Dense osmiophilic bodies, some of which are enclosed by nuclear envelope (heavy arrow), while others have a single membrane around them(arrows). x 21,000.

Figs. 5 and 6. Small membrane-bound spherical bodies, measuring about 130 nm, some of which are free in the m@icleoplasm (arrow), while othersare enclosed by a single membrane (heavy arrows). NP, nuclear envelope projection; Cy, cytoplasmic matter within nuclear invagination. x 21,000.

Fig. 7. A nuclear body with characteristic whorl-like formation by fibrillar material. This body has osmiophilic spherical bodies, membranoussegments (M), and some dense amorphous mass. Within the latter are seen small spherical bodies, 45 nm in diameter, with an electron-lucent substancearound them (arrows). Heavy arrow, granular chromatin matter. One membranous segment appears free in the nucleoplasm. x 42,000.

Fig. 8. A membrane-bound spherical body measuring approximately 120 nm within a nuclear body. x 42,000.

Fig. 9. Portion of a cell from a human neunilemoma cell culture exhibiting margination of chromatin. Arrows, tubuloreticular structures in thecytoplasm. N, nucleus. x 7,500.

Fig. 10. Tubuloreticular structures in Fig. 9 at a higher magnification (arrows). N, nucleus. x 30,000.Fig. 11. An area that appeared to be a remnant of a degenerated nucleus in a culture of human neunilemoma contains spherical bodies, I30 to 230 nm

in diameter. x 9,000.

Figs. 12 and 13. Spherical bodies similar to those in Fig. 11 exhibit bilamellar profiles and spikes on their surfaces at higher magnifications. x78,000.

Fig. 14. For comparison, spikes (arrows) on envelopes of HSV type I and on membranes which may be involved in virus maturation. Othercytoplasmic membranes do not have spikes (heavy arrows). x 78,000.

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1975;35:2000-2006. Cancer Res   Satish Chandra, Michael J. Jerva and Jack D. Clemis  NucleiUltrastructural Characteristics of Human Neurilemoma Cell

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