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Isolation of a C-type virus from mouse myeloma cells
Europ. d. Cancer Vol. 8, pp. 363-364. Pergamon Press 1972. Printed in Great Britain
Letter
to
the Editor
Isolation of a C-type Virus from Mouse Myeloma Cells ABRAHAM KARPAS and JOHN CAWLEY
Department of Medicine, University of Cambridge, Cambridge, England PLASMA cell tumours producing immunoglobulin in the inbred BALB/e strain of mice can be induced experimentally [1]. Typical myeloma has been produced following the administration of, for example, intraperitoneal (i.p.) implantation of solid materials, such as plastic diffusion chambers, plastic shavings, and circular discs of polymethylmethacrylate [2, 3], and by i.p. injections of white mineral oils [1, 4]. Several papers described the presence of A-type particles in the cytoplasm of the pathological cells [5, 6]. These particles were found recently in tissues of normal BALB/c mice [7]. The virus-like A-type particles were reported to be located singly or in clusters within cisternae of the endoplasmic reticulum [6, 7]. Recently it has been reported that immunoglobulin-synthesizing mouse myeloma cells produce an RNA-containing virus which has both an RNA base composition similar to that of other murine leukaemia viral RNA and the group specific antigen of the murine leukaemia-sarcoma group [8]. Ultrastructural studies of the myeloma cells have revealed extra-cellular C-type particles in addition to the intracisternal A-type particles. However, attempts to demonstrate biological activity of this C-type virus both in vitro with a variety of tissue culture lines and in vivo with mice and rats, gave consistently negative results [8]. The successful in vitro isolation of a biologically active C-type virus in normal mouse fibroblasts of the 3T3 cell line is described in the following report.
For the isolation of the C-type virus we used the P-3 plasma cells which are a cloned line (originated by Drs. K. Horibata and M. Cohn) derived from the BALB/c strain of mice and known to produce IgG and to contain viral particles [8]. The P-3 cells were cultured in Dulbecco's modified Eagle's medium (DMM) supplemented with 10 % horse serum. Some of the P-3 cells were fixed in 1.5 % glutaraldehyde for electron microscopy. Other P-3 cells were co-cultivated with 3T3 cells (mouse embryo fibroblast line) and the tissue culture fluid from the mixed culture was passed through a 0.45 # millipore filter, and this filtrate was used to infect 3T3 cultures. Both the infected 3T3 cells and non-infected control ceils were trypsinized and subcultured every 10 days. Following the fifth passage infected and control 3T3 cells were seeded in 50 m m plastic plates and incubated in air with 5 % CO2 at 37°C. After 24 hr 25 #g/ml DEAE-D was added in order to render the cells more susceptible to viral infection [9]. Following the DEAE-D treatment a suspension of the Harvey strain of murine sarcoma virus (MSV-H) was inoculated into infected 3T3 and control cells. After incubation of I hr D M M containing 5 % calf serum was added to the plates and the cultures were placed in the CO2 incubator. The control 3T3 cells infected by M S V - H developed within 4 days numerous foci of transformed cells characteristic of the MSV. In contrast, none of the 3T3 cultures which had been infected earlier with P - 3 - - 3 T 3 co-cultivation :filtrate developed any foci of cell transformation. This observation was repeated three times. The resistance of the infected 3T3 cells to super-
Accepted 17 November 1971.
363
364
Letter to the Editor
infection by M S V is in line with previous observations which indicated that mouse embryo fibroblasts previously infected with any of the murine leukaemia viruses become resistant to super-infection and transformation by the M S V [10]. It suggested therefore that a leukaemia type virus replicated in the 3T3 cells. In order to confirm that a replicating C-type virus is responsible for the resistance of 3T3 cells to infection by MSV, the myeloma virus infected cells and their non-infected control 3T3 cells were processed for electron microscopic examination. Examination of the ultrastructure of the infected 3T3 cells, showed that the cells produce a very large number of mature C-type particles, 100m# in diam. (Fig. 1) thus confirming the biological observation. The virus particles were seen only outside the cell membrane. In contrast, the control 3T3 cells contained no virus particles. It was interesting to note that A-type particles were not detected in any of the 3T3 cells which produced C-type virus, whereas the original myeloma cells (P-3) contained numerous intracisternal A-particles 70 m/~ in diam. similar to
the particles described earlier, and C-type extracellu]ar particles (Fig. 2) which were much less numerous than in the infected 3T3 cells. Tile C-type virus is probably the one which infected and was readily replicating in the 3T3 cells. The A-type particles evidently do not "infect" or replicate in the 3T3 cells. This is in agreement with earlier work which failed to demonstrate any biological activity associatep with the A-particle [6]. The isolation of the C-type virus in 3T3 cells and its massive replication in these cells will allow the harvesting of high concentrations of virus; such preparations may allow experiments to induce myelomatosis in mice. The repeated observations that a wide range of carcinogens can induce the same type of malignancy in mice may suggest that these substances activate the "switched off" vertically transmitted C-type virus. Experimental evidence in support of the hypothesis of cocarcinogenesis of chemical or physical agents together with C-type viruses has been accumulated in recent years [11]. Therefore a similar mechanism of co-carcinogenesis may also operate in the induction o f m y e l o m a in mice.
A©lmowledgments--We thank Dr. C. Milstein for the supply of P-3 cells, and Professor F. G. J. Hayhoe for his helpful comments. The work was supported by
the Leukaemia Research Fund (U.K.). John Cawley gratefully acknowledges the support of the Medical Research Council.
REFERENCES 1. M. POTTER, The plasma cell tumours and myeloma proteins of mice. Methods in Cancer Research (Edited by H. BuscH), 2, 105 (1967). 2. R. M. MERWIN and G. H. ALOmE, Induction of plasma-cell neoplasms and fibrosarcomas in Balb/c mice carrying diffusion chambers. Proc. Soc. exp. Biol. 101, 437 (1959). 3. R. M. MERWXN and L. W. REDMON,Induction of plasma cell tumours and sarcomas in mice by diffusion chambers placed in the peritoneal cavity, or. nat. Cancer Inst. 31, 997 (1963). 4. M. POTTER and C. R. BovcE, Induction of plasma-cell neoplasms in strain Balb/c mice with mineral oil and mineral oil adjuvants. Nature 193, 1086 (1962). 5. A.J. DALTON, M. POTTERand R. M. MERWm, Some ultrastructural characteristics of a series of primary and transplanted plasma-cell tumours of the mouse. J. nat. Cancer Inst. 26, 1221 (1961). 6. E.L. KUFV, N. A. WIVEL and K. K. LUEDER, The extraction of intracisternal A-particles from a mouse plasma cell tumour. Cancer Res. 28, 2137 (1968). 7. N . A . WXVELand G. H. SMITH, Distribution of intracisternal A-particles in a variety of normal and neoplastic mouse tissues. Int. J. Cancer 7, 167 (1971). 8. J. WATSON,P. RALPH,S. SARKARand M. COHN,Leukaemia viruses associated with mouse myeloma cells. Proc. nat. Acad. Sci. (Wash). 66, 344 (1970). 9. K . D . SOMERSand W. H. KIRSTEN,Focus formation by murine sarcoma virus enhancement by DEAE-Dextran. Virology 36~ 155 (1968). 10. P.J. FISGHINGERand T. E. O'CoNNoR, Tissue culture assay of helper activity of murine leukaemia virus for murine sarcoma virus. J. nat. Cancer Inst. 40~ 1199 (1968). 11. R . J . HUE~NER and G. J. TODARO, Oncogenesis of RNA tumour viruses as determinants of cancer. Proc. nat. Acad Sci. (Wash.) 64, 1087 (1969).
Fig. 1. Extra~ellular C-type virus particles in 3T3 culture ( × 48,000). Inset ×90,000.
Fig. 2. P-3 cells containing intracisternal A-type particles in the cytoplasm and C-type particles outside the cell membrane ( × 37,000).
(to lace page 364)
Letter
to
the Editor
Isolation of a C-type Virus from Mouse Myeloma Cells ABRAHAM KARPAS and JOHN CAWLEY
Department of Medicine, University of Cambridge, Cambridge, England PLASMA cell tumours producing immunoglobulin in the inbred BALB/e strain of mice can be induced experimentally [1]. Typical myeloma has been produced following the administration of, for example, intraperitoneal (i.p.) implantation of solid materials, such as plastic diffusion chambers, plastic shavings, and circular discs of polymethylmethacrylate [2, 3], and by i.p. injections of white mineral oils [1, 4]. Several papers described the presence of A-type particles in the cytoplasm of the pathological cells [5, 6]. These particles were found recently in tissues of normal BALB/c mice [7]. The virus-like A-type particles were reported to be located singly or in clusters within cisternae of the endoplasmic reticulum [6, 7]. Recently it has been reported that immunoglobulin-synthesizing mouse myeloma cells produce an RNA-containing virus which has both an RNA base composition similar to that of other murine leukaemia viral RNA and the group specific antigen of the murine leukaemia-sarcoma group [8]. Ultrastructural studies of the myeloma cells have revealed extra-cellular C-type particles in addition to the intracisternal A-type particles. However, attempts to demonstrate biological activity of this C-type virus both in vitro with a variety of tissue culture lines and in vivo with mice and rats, gave consistently negative results [8]. The successful in vitro isolation of a biologically active C-type virus in normal mouse fibroblasts of the 3T3 cell line is described in the following report.
For the isolation of the C-type virus we used the P-3 plasma cells which are a cloned line (originated by Drs. K. Horibata and M. Cohn) derived from the BALB/c strain of mice and known to produce IgG and to contain viral particles [8]. The P-3 cells were cultured in Dulbecco's modified Eagle's medium (DMM) supplemented with 10 % horse serum. Some of the P-3 cells were fixed in 1.5 % glutaraldehyde for electron microscopy. Other P-3 cells were co-cultivated with 3T3 cells (mouse embryo fibroblast line) and the tissue culture fluid from the mixed culture was passed through a 0.45 # millipore filter, and this filtrate was used to infect 3T3 cultures. Both the infected 3T3 cells and non-infected control ceils were trypsinized and subcultured every 10 days. Following the fifth passage infected and control 3T3 cells were seeded in 50 m m plastic plates and incubated in air with 5 % CO2 at 37°C. After 24 hr 25 #g/ml DEAE-D was added in order to render the cells more susceptible to viral infection [9]. Following the DEAE-D treatment a suspension of the Harvey strain of murine sarcoma virus (MSV-H) was inoculated into infected 3T3 and control cells. After incubation of I hr D M M containing 5 % calf serum was added to the plates and the cultures were placed in the CO2 incubator. The control 3T3 cells infected by M S V - H developed within 4 days numerous foci of transformed cells characteristic of the MSV. In contrast, none of the 3T3 cultures which had been infected earlier with P - 3 - - 3 T 3 co-cultivation :filtrate developed any foci of cell transformation. This observation was repeated three times. The resistance of the infected 3T3 cells to super-
Accepted 17 November 1971.
363
364
Letter to the Editor
infection by M S V is in line with previous observations which indicated that mouse embryo fibroblasts previously infected with any of the murine leukaemia viruses become resistant to super-infection and transformation by the M S V [10]. It suggested therefore that a leukaemia type virus replicated in the 3T3 cells. In order to confirm that a replicating C-type virus is responsible for the resistance of 3T3 cells to infection by MSV, the myeloma virus infected cells and their non-infected control 3T3 cells were processed for electron microscopic examination. Examination of the ultrastructure of the infected 3T3 cells, showed that the cells produce a very large number of mature C-type particles, 100m# in diam. (Fig. 1) thus confirming the biological observation. The virus particles were seen only outside the cell membrane. In contrast, the control 3T3 cells contained no virus particles. It was interesting to note that A-type particles were not detected in any of the 3T3 cells which produced C-type virus, whereas the original myeloma cells (P-3) contained numerous intracisternal A-particles 70 m/~ in diam. similar to
the particles described earlier, and C-type extracellu]ar particles (Fig. 2) which were much less numerous than in the infected 3T3 cells. Tile C-type virus is probably the one which infected and was readily replicating in the 3T3 cells. The A-type particles evidently do not "infect" or replicate in the 3T3 cells. This is in agreement with earlier work which failed to demonstrate any biological activity associatep with the A-particle [6]. The isolation of the C-type virus in 3T3 cells and its massive replication in these cells will allow the harvesting of high concentrations of virus; such preparations may allow experiments to induce myelomatosis in mice. The repeated observations that a wide range of carcinogens can induce the same type of malignancy in mice may suggest that these substances activate the "switched off" vertically transmitted C-type virus. Experimental evidence in support of the hypothesis of cocarcinogenesis of chemical or physical agents together with C-type viruses has been accumulated in recent years [11]. Therefore a similar mechanism of co-carcinogenesis may also operate in the induction o f m y e l o m a in mice.
A©lmowledgments--We thank Dr. C. Milstein for the supply of P-3 cells, and Professor F. G. J. Hayhoe for his helpful comments. The work was supported by
the Leukaemia Research Fund (U.K.). John Cawley gratefully acknowledges the support of the Medical Research Council.
REFERENCES 1. M. POTTER, The plasma cell tumours and myeloma proteins of mice. Methods in Cancer Research (Edited by H. BuscH), 2, 105 (1967). 2. R. M. MERWIN and G. H. ALOmE, Induction of plasma-cell neoplasms and fibrosarcomas in Balb/c mice carrying diffusion chambers. Proc. Soc. exp. Biol. 101, 437 (1959). 3. R. M. MERWXN and L. W. REDMON,Induction of plasma cell tumours and sarcomas in mice by diffusion chambers placed in the peritoneal cavity, or. nat. Cancer Inst. 31, 997 (1963). 4. M. POTTER and C. R. BovcE, Induction of plasma-cell neoplasms in strain Balb/c mice with mineral oil and mineral oil adjuvants. Nature 193, 1086 (1962). 5. A.J. DALTON, M. POTTERand R. M. MERWm, Some ultrastructural characteristics of a series of primary and transplanted plasma-cell tumours of the mouse. J. nat. Cancer Inst. 26, 1221 (1961). 6. E.L. KUFV, N. A. WIVEL and K. K. LUEDER, The extraction of intracisternal A-particles from a mouse plasma cell tumour. Cancer Res. 28, 2137 (1968). 7. N . A . WXVELand G. H. SMITH, Distribution of intracisternal A-particles in a variety of normal and neoplastic mouse tissues. Int. J. Cancer 7, 167 (1971). 8. J. WATSON,P. RALPH,S. SARKARand M. COHN,Leukaemia viruses associated with mouse myeloma cells. Proc. nat. Acad. Sci. (Wash). 66, 344 (1970). 9. K . D . SOMERSand W. H. KIRSTEN,Focus formation by murine sarcoma virus enhancement by DEAE-Dextran. Virology 36~ 155 (1968). 10. P.J. FISGHINGERand T. E. O'CoNNoR, Tissue culture assay of helper activity of murine leukaemia virus for murine sarcoma virus. J. nat. Cancer Inst. 40~ 1199 (1968). 11. R . J . HUE~NER and G. J. TODARO, Oncogenesis of RNA tumour viruses as determinants of cancer. Proc. nat. Acad Sci. (Wash.) 64, 1087 (1969).
Fig. 1. Extra~ellular C-type virus particles in 3T3 culture ( × 48,000). Inset ×90,000.
Fig. 2. P-3 cells containing intracisternal A-type particles in the cytoplasm and C-type particles outside the cell membrane ( × 37,000).
(to lace page 364)