INFECTIOUS MURINE TYPE-C VIRUSES RELEASED FROM HUMAN CANCER CELLS TRANSPLANTED INTO NUDE MICE

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1 INFECTIOUS MURINE TYPE-C VIRUSES RELEASED FROM HUMAN CANCER CELLS TRANSPLANTED INTO NUDE MICE Toshimitsu SUZUKI,*1 Kazuyoshi YANAGIHARA,*2 Koichi YOSHIDA,*2 Tsutomu SEIDO,*1 Norito KUGA,*3 Yukio SHIMOSATO,*2 and Shoichi OBOSHI*1 Department of Pathology, Niigata University School of Medicine,*1 Pathology Division, National Cancer Center Research Institute,*2 and Department of Ophthalmology, Hiroshima University School of Medicine*3 Type-C virus particles were revealed by electron microscope in 6 of 9 tumors of cultured and biopsied human cancers heterotransplanted into nude mice. Some tumors in nude mice were explanted for in vitro cultivation. The virus particles were also found in the cultures derived from the virus-positive tumors. They were mostly found extracellularly, but the particles in budding process were also encountered frequently. Cytological study and karyotype analysis of the cultured cells proved these virus-releasing cells as of human origin. From the close correlation between the statistical virus counts and the complement fixation titers for murine gs antigen of the tumors and their cultures, these viruses propagated in human cancer cells were confirmed to be infectious viruses of nude mouse origin. The virus replicating in human cancer cells was readily infected in some of innocent human cancer cells by co-cultivation. It is to be emphasized that infection of animal endogenous viruses on heterotransplanted human cancer cells is a bothersome contamination for human cancer research, especially when searching for a human tumor virus candidate. Congenitally athymic nude mice accept various cultured human cancer cells and human cancer tissues.11) Some tumors developed in nude mice after heterotransplantation yielded type-c particles.5) Murine nontransforming oncornaviruses can be divided on the basis of their host range into those which are restricted in mouse cells (ecotropic), those which grow well in cells of many other species (xenotropic), and those which infect mouse cells and cells of other species (amphotropic).6,12,18) The xenotropic viruses have a wide host range, including human beings. Therefore human tumor cells heterotransplanted into mice can be contaminated with murine endogenous viruses. The substantial effect of murine endogenous virus infection on the transplanted tumor, however, have not been clarified. In our study the particles observed in human cancer cells which were inoculated into nude mice had murine speciesspecific gs antigen, and these were readily infected to some of virus-free human tumor cells by co-cultivation. Viral contamination of heterotransplanted human cancer cells in nude mice is discussed. MATERIALS AND METHODS Cells and Tumors The cultured human cancer cells and human tumors used for transplantation into nude mice have been described previously.11) In this study, a biopsy meterial, LP-4, obtained from liposarcoma was added. The culture and inoculation methods were also described in our previous paper.11) A nude mouse 99

2 T. SUZUKI, ET AL. embryo (NME) cells were cultured from an 18- day embryo. A part of inoculum was immersed in 10% dimethyl sulfoxide, frozen in liquid nitrogen, and stored. After 10 months, the cells were thawed and cultured. This culture was named NME-F. Animals Eight-week-old nude mice, BALB/c origin, supplied from Central Institute of Experimental Animals, Kawasaki, were employed. These were raised under specific pathogen-free conditions. Antiserum A rabbit antiserum against speciesspecific gs-1 antigen of MuLV9) was kindly supplied by Dr. S. Hino, Institute of Medical Science, University of Tokyo. It was absorbed completely with fetal calf serum before use. Specificity of the antiserum was checked by some control human cultured cells which had not been passaged through any animals. Many of them showed a negative result but a few of them reacted positive- ment fixation titer was regarded as significantly positive. Complement Fixation Test Tumors developed in nude mice were homogenized and pelletized at 3000rpm for 15min and 0.1ml of precipitate was suspended in equal volume of saline. Cultured cells were washed 3 times with phosphate-buffered solution, ph 7.4, and centrifuged at 1000 rpm for 10min. A suspension of 0.1ml of cell pack in equal volume of saline was subjected to 5 cycles of freezing and thawing, and freezestored at -80 until use. Complement fixation (CF) test was done according to the method described by Hino et al.9,10) Co-cultivation The NB-1-nu cells, which had passed through nude mouse and confirmed to be virus-yielding cells were trypsinized and centrifuged after irradiation of 5000rad of X-ray. The aortic endothels of the Donryu rat origin, a rat type-c producer,14) were treated in the same way as the NB-1-nu cells. The NME-F cells, which were thawed and subcultured for the 8th passage, could not be checked for virus production but used for co-cultivation after the same treatment. The centrifuged precipitates of the above-mentioned cells were placed on NB-1 and HPL cells. At the 5th subculture, the cells were studied with electron microscope. Electron Microscopy and Statistical Virus Counts The tumor and the cells were fixed for 1hr with cold 1% glutaraldehyde, washed with Sorensen's buffer, postfixed for 30min in 1% OsO4, and embedded in epoxy resin (Epon 812). Thin sections were stained with uranyl acetate and lead citrate before examination in a Hitachi HU-11E or HS-8 electron microscope. The statistical virus counts were made on the number of type-c particles in the section of 100 consecutive cells. RESULTS Tables I to III summarize the results. Nine tumors developed in nude mice were examined with an electron microscope. Six tumors were produced by the inoculation of cultured cells (SCH, MOLT, NB-1, PC-5, PC-6, and HPL lines) and others of biopsied materials (YT-nu, LP-1, and LP-4). In the tumors produced by PC-5, PC-6, HPL, and NB-1 cells, type-c particles were revealed by electron microscopy in varying numbers. In the tumors produced by LP-4 and YT-nu, type-c particles were also observed. Mostly they were found extracellularly and appeared to be similar to mature and immature murine leukemia virus (Photo 1), and particles in the budding process were encountered frequently (Photo 2). The statistical counts of virus particles in the section of 100 consecutive cells are listed in Table I. In the tumors produced by SCH and MOLT cells, or LP-1, vigorous efforts to search for a type-c particles were in vain. The tumor homogenates were examined by the CF tests for murine type-c virus gs-1 antigen. Those that released type-c viruses showed high CF titers. As shown in Table I, little discrepancies were noted between the results obtained from statistical virus counts and CF tests. The results of CF test of the tumor homogenates do not reflect the genuine titer, because their involvement of the host cells was not eliminated completely, and it is not easy to identify the virus-yielding cells proliferating in the nude mice as human origin by electron microscopy. Some tumors in the nude mice were explanted and reestablished as in vitro cell cultures. After identification of the newly established cell lines as human origin, the statistical virus counts and CF tests were employed again. 100 Gann

3 INFECTIOUS VIRUS OF NUDE MOUSE Table I. Statistical Virus Count and gs Antigen Titer of Tumor Transplanted into Nude Mice Table II. Statistical Virus Count and gs Antigen Titer of Cultured Cells from Nude Mouse Tumors These tests were made after the tumor cells were identified as human origin since they were contaminated with nude mouse cells. The SCH cells derived from choriocarcinoma of the stomach consisted of mononuclear and syncytial cells. As the syncytial cells have intracytoplasmic desmosomes, their identification is not difficult. The SCH cells yielded no virus particles either in vivo or in vitro. Back-cultured MOLT cells also remained free of overt type-c virus expression. The NB-1 cells derived from neuroblastoma have neurosecretory granules in their cytoplasm. Photo 3 shows the virus budding from the cell which has a neurosecretory granule in its cytoplasm. The YT-nu tumor produced by the inoculation of the surgical material of neuroblastoma was explanted for in vitro cultivation. The cells propagated readily in culture and were named YT-nu line. Its chromosome number was 49 and the karyotype was that of a human. Coincidental chromosome analysis of nude mouse bone marrow cells revealed that the mode of chromosome was 40 and all of them were acrocentric. The in vitro-cultured YT-nu cells also released type-c particles. Therefore, these cultures have both neurosecretory granules and virus particles in the same cells (Photo 4). The CF titer and statistical virus counts of the cultured cells that had passed through nude mice were similar to those of tumor homogenates (Table II). A few virus particles were observed in the spleen cells of a 23-weekold nude mouse (Table I, Photo 5). The NB-1 cells which had been passaged through nude mice and back-cultured began to yield numerous type-c viruses and were named NB-1-nu cells. The NB-1 or HPL cells were co-cultivated with NB-1-nu cells which had received 5000rad X-ray irradiation previously. The co-cultured NB-1 and HPL cells readily released type-c particles. The statistical virus counts were 6 and 18, respectively. The NB-1 or HPL cells were also co-cultivated with NME-F cells which were not confirmed to be a virus producer or not. In this case, no virus particles were observed in the NB-1 or HPL cells (Table III). 68(1)

4 Table III. Virus Infection from Producer Cells to Non-producers by in vitro Coculture In the tumor produced by NB-1 cells and in NB-1-nu cultured cells, atypical-shaped viruses were observed. They were filamentous or cylindrical, having an outer diameter of approximately 80 to 90nm and the longest T. SUZUKI, ET AL. viral particles were located intracisternally and associated with typical budding particles (Photo 6). This type of virus, however, was not found in either other tumors or cultured cells. DISCUSSION Six cultured cell lines and three surgical materials of human cancer were transplanted into nude mice and each of them developed tumor mass. Four tumors derived from the former and 2 tumors from the latter materials were found to generate type-c particles in various numbers by electron microscopy. The CF titers using murine type-c gs-1 antigen of the tumor homogenates showed rough mutual relationship to the statistical virus counts of the tumor cells. Four out of nine heterotransplants which grew in the nude mice were explanted for in vitro cultivation. After each of them was confirmed to be of human origin by karyotype analysis, the CF tests and statistical virus counts were made. The CF titers were more closely correlated to the virus counts, since contaminated nude mouse cells were more depleted in in vitro than in in vivo experiment. From these results, the type-c viruses found in heterotransplanted human cancer cells were concluded as murine endogenous viruses of nude mouse origin. The possibility of laboratory contamination with common murine leukemia virus can be excluded, since in our laboratory none of the cell lines of mouse origin is maintained. The nude mice which we used in the experiment were on the genetic background of the BALB/c. It has been well known that a mouse harbors at least two types of endogenous virus; BALB virus-1 and BALB virus-2.1) The BALB virus-1, which can propagate slightly in the rat cells, grows mostly in the NIH Swiss mouse cells and can therefore be called an N-tropic virus. The other endogenous virus, BALB virus-2, however, cannot propagate in the mouse cells but in rat or human cells (xenotropic). Moreover, the virus grows more preferentially in human cells than in rat cells.17) Taking the tropism of the BALB/c endogenous virus (es) into consideration, the type- C viruses observed in our experiment might be xenotropic or amphotropic, although the tropism had not been checked. Recovery of the type-c viruses from heterotransplanted human cancers into the conditioned mouse2,18) or nude mouse16) has been described in several papers, and the virus is regarded as the murine endogenous virus. The mouse endogenous viruses were found even in the cell line derived from the NIH Swiss, which had been thought to be virusfree, and the endogenous viruses included the virus with xenotropism.12) The nude mice, used widely at present in cancer or immunological research, are genetically of the BALB/c origin or hybrid of the BALB/c and the NIH Swiss.16) Therefore, the BALB/c nude mouse may harbor at least murine type-c viruses of two categories and the NIH nude, as expected 102 Gann

5 INFECTIOUS VIRUS OF NUDE MOUSE from its genetic background, of three categories; N-tropic, B-tropic, and X-tropic. Heterotransplantation of human malignancies into the athymic nude mice is a convenient and useful tool for human cancer research but, after all, a bothersome contamination of transplanted human cells with infectious murine endogenous type-c viruses should be inevitable in most cases, since we have not any virus-free nude mice available. The murine type-c virus propagated in the human cancer cells can be infected to other virus-free permissive human cells in vitro. Therefore, we should handle the murine type-c virus producers such as human tumors passaged through the mouse or transplantable mouse tumors13) with great care to avoid laboratory hazards. An established cell line from the transplanted tumors or reestablished one from the animal passage will become persistent or at least potential producers of the animal endogenous viruses. To avoid confusion, the mark indicating the host animal should be added to the cell line name, such as YT-nu, which means the YT-cell line (human neuroblastoma) established through heterotransplantation into the nude mice. The substantial influence of murine endogenous virus infection on human cancer cells has not been clarified. Recently it has been reported that human malignant lymphoma cells infected with murine endogenous viruses after heterotransplantation could more readily be cultured than that explanted from the primary human lymphoma,5) which is notoriously difficult to cultivate. The possibility that they enhance the growth potential of human lymphoma cells still remains. In general, however, the heterotransplants in the nude mice or conditioned animals can be more easily cultured than the primary explants for culture. A cylindrical filamentous form of the virus found in our study was first described by Dalton et al.3) in the experiment using Moloney leukemia virus. Later, Dmochowsky et al.,4) Fujinaga et al.,7) and Hall et al.8) observed such atypical viruses. About its significance, Dmochowsky stated that it might be regarded as a non-segmented spherical form of the virus. Orenstein and Weinstein,15) however, alleged that this atypical form would be due to aberration in virus assembly and maturation, since the number of this atypicalformed virus increased after BUdR treatment. We considered that the filamentous form of the virus observed in NB-1 cells would be produced by an abnormal viral replication, because no segmentation was observed in the filamentous viruses. This work was supported in part by a Grantin-Aid for Cancer Research from the Ministry of Education, Science and Culture, and from the Ministry of Health and Welfare. (Received October 1, 1976) REFERENCES 103

6 T. SUZUKI, ET AL. Photo 1. Type-C particles, mature and immature, in a nude mouse-grown PC-6 tumor EXPLANATION OF PLATES Photo 2. Budding of the type-c particle in a nude mouse tumor derived from NB-1 (2a) Photo 3. A neurosecretory granule (arrow) and a budding type-c particle (double arrows) in the same cell of a nude mouse-grown NB-1 Photo 4. Both neurosecretory granule (arrow) and the virus budding (double arrows) in the Photo 5. Type-C particles of a normal nude Photo 6. Filamentous forms of the virus, associated with the budding particle (arrow) but 104 Gann

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