In order to study the viral etiology of human brain
tumors, attempts were made to isolate cytopathogenic agents from human brain tumors by the tissue culture of tumor tissues and by the mixed culture of tumor tissues with HeLa cells. Five glioblastomas, a mixed form of glioblastoma and fibrosarcoma, two astrocytomas, two ependymomas, two meningiomas, an oligodendroglioma, a spongioblastoma polare and a choroid plexus papilloma were studied. In the tissue culture, besides the cells which appeared to be the tumor parenchymal cells, varying amounts of fibroblastic cells appeared in all the tumors tested and they increased with the prolongation of the
culture period. In any of the tumors tested, no cytopathogenic agents were detected by either the culture of tumor tissues or the mixed culture of tumor tissues with HeLa cells. From the virological point of view, the significance of these negative results was discussed.</p

Yabe, Yoshiro

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 28, Issue 4 1974 Article 2AUGUST 1974Attempts to isolate virus from human braintumorsYoshiro Yabe∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Attempts to isolate virus from human braintumors∗Yoshiro YabeAbstractIn order to study the viral etiology of human brain tumors, attempts were made to isolatecytopathogenic agents from human brain tumors by the tissue culture of tumor tissues and by themixed culture of tumor tissues with HeLa cells. Five glioblastomas, a mixed form of glioblas-toma and fibrosarcoma, two astrocytomas, two ependymomas, two meningiomas, an oligoden-droglioma, a spongioblastoma polare and a choroid plexus papilloma were studied. In the tissueculture, besides the cells which appeared to be the tumor parenchymal cells, varying amounts offibroblastic cells appeared in all the tumors tested and they increased with the prolongation of theculture period. In any of the tumors tested, no cytopathogenic agents were detected by either theculture of tumor tissues or the mixed culture of tumor tissues with HeLa cells. From the virologicalpoint of view, the significance of these negative results was discussed.∗PMID: 4374049 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 28,247-252 (1974)ATTEMPTS TO ISOLATE VIRUS FROM HUMANBRAIN TUMORSYoshiro YABEDepartment flf Virology, Cancer Institute, Okayma University Medical School,Okayama, Japan (Director: Prof. Y. Yabe)Rceived for publication, January 19, 1974Abstract: In order to study the viral etiology of human braintumors, attempts were made to isolate cytopathogenic agents fromhuman brain tumors by the tissue culture of tumor tissues and by themixed culture of tumor tissues with HeLa cells. Five glioblastomas,a mixed form of glioblastoma and fibrosarcoma, two astrocytomas,two ependymomas, two meningiomas, an oligodendroglioma, a spon-gioblastoma polare and a choroid plexus papilloma were studied. Inthe tissue culture, besides the cells which appeared to be the tumorparenchymal cells, varying amounts of fibroblastic cells appeared inall the tumors tested and they increased with the prolongation of theculture period. In any of the tumors tested, no cytopathogenic agentswere detected by either the culture of tumor tissues or the mixedculture of tumor tissues with HeLa cells. From the virological pointof view, the significance of these negative results was discussed.The brain has a high sensitivity to many viruses (I). When inoculatedintracranially into animals, as reviewed by IKUTA and KUMANISHI (2), manytumor viruses induce brain tumors. As for human brain tumors, there areseveral reports on the electron microscopic observation of virus-like struc-tures, though some of them are atypical for virus particles, in tumor tissuesor in tissue culture cells of tumors (3-10).It is known, on the other hand, that several types of human adenovirusinduce tumors in laboratory animals (11, 12). Of the various opinions onthe origin of the tumors induced by human adenoviruses, the neuroectodermalorigin has been proposed by OcAWA et at. (13, 14). The relationship betweenhuman adenoviruses and human tumors has been studied by the attempts toisolate adenoviruses from human tumors or cancer patients (15-17), to findT-antigen in human tumors and T-antibody in cancer patient sera (18-23) andto find adenovirus-specific mRNA in human tumors (24, 25). No definiteresults have been so far obtained, but MALMGREN et at. reported that braintumors were one of the tumor types which seemed most often to be associatedwith adenovirus-T-antibody-positive sera (21). These results suggest that atleast some of human brain tumors might be of adenovirus origin. It thusappeared significant to attempt the isolation of Viruses, particularly adeno-2471Yabe: Attempts to isolate virus from human brain tumorsProduced by The Berkeley Electronic Press, 1974248 Y. YABEviruses, from human brain tumors. In the present study, the author tried toisolate viruses from human brain tumors by the tissue culture of tumorsand by the mixed culture of tumor tissues with BeLa cells.MATERIALS AND METHODSBrain tumors: A part of human brain tumors removed surgically was im-mediately put in an ice box and transferred to our laboratory.Tissue culture media: Earle's balanced salt solution containing 0.1% yeastextract and 0.5% lactalbumin hydrolysate (YLE) was prepared in our labora-tory, and medium 199 was obtained commercially. Just before use, these mediawere supplemented with heat-inactivated bovine serum of varying concentra-tions.HeLa cells: Wild type BeLa cells maintained in our laboratory were used.BeLa cells were cultured in YLE with 10% bovine serum.Culture of tumor tissues: Tumor tissues were minced with sharp scissors,trypsinized and suspended in YLE or medium 199 supplemented with 50%bovine serum. The suspension was poured at 1 ml/tube into twenty test tubes,of which four contained coverslips for cell staining, stoppered and placed inan incubator of 37°C.Mixed culture with HeLa cells: After 3-4 weeks' incubation, tumor tissueculture tubes were divided into two groups. The first group was maintainedas before without any treatment except for decreasing the serum content ofthe medium to lO%-tumor group. In the second group, 104 BeLa cells pertube were planted onto the tumor cell cultures and maintained in the sameway as the first group-tumor-HeLa-mixed group. As the control to thesecond group, five test tubes were planted with 104 HeLa cells per tube-HeLagroup. All these groups were further maintained for 35-37 days and thenstored at - 70°C.Tissue culture passage: Cells of the tumor and the tumor-HeLa-mixedgroups stored for 1-7 days at - 70°C were thawed, mixed and inoculated intothree BeLa cell tubes (1 ml/tube). On the next day, the medium was changedwith YLE containing 2% bovine serum, and maintained for 14-17 days.Medium change and observation: Throughout all these tissue culture pro-cesses, medium change and light microscopic observation were done at leasttwice a week. Everyone or two weeks, cells cultured on coverslips were fixedin formalin, stained in hematoxylin and eosin, and observed for the cyto-pathogenic effect.RESULTSAs shown in Table I, five glioblastomas, a mixed form of glioblastomaand fibrosarcoma, two astrocytomas, two ependymomas, two meningiomas,an oligodendroglioma, a spongioblastoma polare and a.choroid plexus papil-loma were studied. Varying degrees of cell growth were observed in all2Acta Medica Okayama, Vol. 28 [1974], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss4/2Virus and Human Brain Tumors 249TABLE 1 ATTEMPTS TO ISOLATE VIRUS FROM HUMAN BRAIN TUMORSPatient Viral cytopathogenic effect inTumor Cell Tumor Mixed culture PassageAge Sex Growthl > tissue of tumor and ontoculture HeLa cells HeLa cells 2 )30 6 Glioblastoma +++40 6 Glioblastoma +++42 ~ Glioblastoma +++48 ~ Glioblastoma +++60 6 Glioblastoma +++59 6 Mixed form ofglioblastoma and +++fibrosarcoma4 6 Astrocytoma ++33 ~ Astrocytoma ++28 6 Ependymoma +29 6 Ependymoma ++48 6 Meningioma ++49 ~ Meningioma ++62 6 Oligodendroglioma ++22 ~ Spongioblastoma polare +69 6 Choroid plexus papilloma ++1) Cell growth in tumor cell cultures at the end of 56-65 days' maintenance. + Cells occupy20-30% of the culture surface, + + 40-50%, + + + over 50%.2) Frozen-thawed suspensions of the tumor tissue culture and the mixed culture of tumor andHeLa cells maintained for 56-65 days were inoculated onto HeLa cells, and observed forthe cytopathogenic effect for additional 14-17 days.these tumors, but it appeared better in glioblastomas than in others. Besidesthe cells which appeared to be the tumor parenchymal cells, varying amountsof fibroblastic cells appeared in all the tumors cultured. With the prolonga-tion of the period of in vitro culture, fibroblastic cells increased, and, aroundthe end of the culture period of 56-65 days, they constituted the majority.In the mixed cultures of tumor tissues and HeLa cells, HeLa cells overgrewtumor cells in the later stage of culture. During the whole period of 56-65days of the primary culture, cells were observed for the cytopathogeniceffect. In all tumors tested, as shown in Table 1, the cytopathogenic effectwas not observed in any of the cultures of tumor tissues or the mixed culturesof tumor tissues with HeLa cells.The cell cultures of these tumors were frozen-thawed, further inoculatedinto HeLa cells and maintained for 14-1 7 days, but no cytopathogenic effectwas observed (Table 1).3Yabe: Attempts to isolate virus from human brain tumorsProduced by The Berkeley Electronic Press, 1974250 Y. YABEDISCUSSIONIsolation of virus from tumor cells does not necessarily mean that thevirus is the causative agent of the tumor. From the virological point of view,however, animal tumors may be divided into four groups: 1) tumors whichusually yield the causative virus, e. g. tumors induced by many RNA tumorviruses and papilloma or pox viruses; 2) tumors which usually do not yieldthe causative virus but yield the virus under such appropriate conditions asin the tissue culture of tumor cells, in the mixed culture of tumor cells withvirus-sensitive indicator cells or in a similar mixed culture in which cells arefused by cell fusion technique, e. g. hamster tumors induced by SV40 (26-28) ;3) tumors which are evidently viral but do not yield the causative virus underthe conditions so far tested, e. g. hamster tumors induced by polyoma virus orhuman adenoviruses (26, 29); and 4) tumors in which no causative relationswith viruses have been detected by the methods available at present.In the present study, only the culture of tumor tissues and the mixedculture of tumor tissues with HeLa cells were done, and the cytopathogeniceffect was taken as the only indication for the presence of virus. Hence thesignificance of the negative results in the present study is very limited, andmore informations will be obtained by further studies by such methods as theuse of human embryonic brain cells for isolation of viruses, the cell fusiontechnique to enhance the production of viruses, inoculation of tumor extractsor tissue culture fluids into the brain of animals, particularly into that of non-human primates, and the electron microscopy of the culured tumor cells.It is known, however, that the tissue culture of adenoids or tonsilsfrequently discloses the latent adenoviruses, showing the specific cytopatho-genic effect in the cultured cells (30). It is also known well that BeLa cellsare sensitive to adenoviruses and show the specific cytopathogenic effect attheir infection (30). Therefore, the negative results in the present study wouldimply at least that these fifteen human brain tumors tested did not harboradenoviral gene rescuable by such conventional methods as employed in thepresent study. McALLISTER et ai. reported the isolation of adenovirus type 1from an astrocytoma by similar methods (1 7). Their experiments to deter-mine the significance of the isolated virus, however, did not indicate whetherthe virus had the etiological significance or whether it was a laboratory con-taminant or a passenger virus in the tissue. SABIN also briefly describednegative results in a similar attempt to isolate viruses from neuroblastomas(31). Our repeated attempts to transform human embryonic brain cells invitro by human adenovirus type 12 were unsuccessful (32).4Acta Medica Okayama, Vol. 28 [1974], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss4/2Virus and Human Brain Tumors 251Acknowledgement: The author is grateful to Prof. S. TANAKA and Prof. A. NISHIMOTO ofour medical school for supplying materials, and to Miss N. KATAOKA fpr preparing manu-script. This work was supported in part by a grant from the Ministry of Education.REFERENCESI. RHODES, A. .1. and van ROOYEN, C. E.: Textbook of Virology, 3rd Ed., p. 69, Williams& Wilkins, Baltimore, U.S.A. 19582. IKUTA, F. and KUMANISHI, T.: Experimental virus-induced brain tumors. Progress in Neuro-pathology 3, 253-334, 19733. KOINov, R.: Crystal bodies in the ultrastructure of multiform glioblastoma. Cancer 20,1181-1187, 19674. VAZQUEZ, J. J. und CERvos-NAVARRO, J.: Intranucleare stabformige Gebilde bei einemOligodendrogliom. Acta neuropath. (Berl.) 13, 289-293, 19695. TANI, E., YAMASHITA, j., TAKEUCHI, J. and HANDA, H.: Polygonal crystalline structuresand crystalline aggregates of cylindrical particles in human glioma. Acta neuropath. (Berl. )13, 324-337, 19696. TANI, E. and AMETANI, T.: Polygonal crystalline structures in human ependymoma cells.Acta neuropath. 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B.: Manifestations of latent viral genetic material in experimentally producedcancers and search for such manifestations in human cancers. Israel }. Med. Sci. 1, 93-103, 196532. Y ABE, Y.: Unpublished observations.6Acta Medica Okayama, Vol. 28 [1974], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss4/2

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