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A glial fibrillary acidic protein (GFA)-containing cell clone derived from mouse cerebella transformed ‘in vitro’ by SV-40
Brain Research, 216 (1981) 455--459 © Elsevier/North-Holland Biomedical Press
455
A glial fibrillary acidic protein (GFA)-containing cell clone derived from mouse cerebella transformed 'in vitro' by SV-40
F. ALLIOT and B. PESSAC* C.N.R.S. E.R. 231 and LN.S.E.R.M. U178, H@ital Broussais, 96 rue Didot, 75674 Paris Cedex 14 (France)
(Accepted March 5th, 1981) Key words: mouse - - cerebellum - - SV-40 - - cell clone - - glial fibrillary acidic protein - - T-antigen
A clonal cell line containing the glial fibrillary acidic protein (GFA) was derived from cultures of 14 day post-natal C57B1 mouse cerebella, transformed by SV-40. All the cells are GFA-positive - even in sparse exponential cultures - - have an astroglial morphology and contain the intranuclear SV40 T-antigen. The glial fibrillary acidic protein (GFA)-containing cell clones which have been reported in the literature were obtained from cultures isolated from chemically induced brain tumorsl,~, 10. This approach suffers from the drawback of not being able to determine the tissue of origin of these tumors. In an attempt to obtain cell lines from the various cell types of cerebellum, we transformed, with SV-40, cultures of mouse cerebellum cells taken at various stages of embryonic and postnatal development 6. Several cultures and clones have been established permanently in this way; in the present paper we confine our description to a GFA-positive clone derived from 14-day post-natal C57B1 mouse cerebella. Mice were sacrificed by decapitation, and cerebella quickly removed. The meninges were dissected out and the cerebella cut into small pieces. The tissue was dissociated by gentle pipetting in Eagle's basal medium (BME). Single cells and clumps were plated in tissue culture dishes coated with poly-L-lysine (10/~g/ml) in B M E q15 % fetal bovine serum (FBS) and immediately infected with about 10 FFU/cell of a wild type SV-40 (LP strain Rh 911, a gift of Dr. V. Defendi). The medium was renewed the following day and every week thereafter. One week after infection, the dishes were covered with confluent cell monolayers. Foci of apparently proliferating cells appeared throughout the culture after 25-30 days. The first subculture was done two months later, at a ratio of 1/3, and cloning started at the third subculture. The cultures were cloned by dilution because initial attempts to clone single isolated cells were unsuccessful. Single cells and small clumps (2-5 cells) dissociated from the culture by pipetting *To whom correspondence should be addressed.
456
Fig. 1. A and B: immunofluorescence, with 1/40 GFA antiserum, of the mouse cerebellum clonal cells K5-5. A, after 4 days and B, after 7 days in culture. The cytoplasm and processes are brightly fluorescent. x 300.
in BME + 20 ~ FBS, were mixed with 5 x 104 cells/ml of the same lethally irradiated culture which served as a feeder. The cell suspension was plated on Costar 'Tissue culture cluster 96' according to the Poisson distribution. This procedure was repeated 5 times over a 2.5 year period. Cell morphology remained heterogeneous, suggesting that the cultures were made up of two or more cell types. Only then were we able to obtain single cell clones by plating very diluted suspensions on coverslip fragments. One of the clonal cell lines tested, named CBl4-SV-4A4K5-5 (referred to in brief as K5-5), contained G F A (detected by indirect immunofluorescence with anti-GFA sera) and could therefore be identified as astroglial. The assay was performed on cells which were grown for 4-8 days on glass coverslips in tissue culture flasks, in BME t 10/o°/FBS in 95 ~ air, 5 ~ CO2, washed 3 times in PBS and fixed by immersion in acetone at - - 2 0 °C for 5 rain. The cells were then successively incubated in normal goat serum (l/6, l0 min), rabbit anti-GFA serum (batch numbers 35 and 75, a gift of Dr. A. Bignami) at a dilution of 1/40 for 120 min, and fluorescein-conjugated goat anti-rabbit immunoglobulin (Nordic Immunological Laboratories, Tilburg, Netherlands; 1/20 for 30 min). All incubations were done at room temperature and were followed by 3 rinses in PBS. The coverslips were mounted on slides in buffered glycerol and viewed with a Leitz epifluorescence microscope with appropriate filters.
457 In sparse cultures, all cells were brightly immunofluorescent and staining was confined to cytoplasm and processes (Fig. 1A, B). Their morphology was reminiscent of that of the other astroglial clones 1,2,10, with a thin rim of cytoplasm around a large nucleus and with few processes. It did not change in the 6 months following the single cell cloning and has remained constant ever since. All the cells were GFA-positive in both the exponential and stationary growth phases but extended more processes in the stationary phase. Although the specificity of these anti-GFA sera, as a marker for astroglial cells in culture, has been established 8, non-immune sera and immune sera not reacting to G F A (like an anti-tetanus toxoid serum), were used as controls, and were negative. Further evidence for the specificity of the anti-GFA sera came from the fact that staining was abolished by preincubation with human G F A (a gift of Dr. C. Jaque) (Fig. 2). The intranuclear SV-40 T antigen 7 was present in all ceils, thus proving that they were actually transformed by SV-40 (Fig. 3). The cells of clone K5-5 grow readily in BME -k 1 0 ~ FBS with a doubling time of about 24 h.
Fig. 2. Immunofluorescenceof K5-5 cells after 4 days in culture, with a 1/40 GFA antiserum absorbed with human GFA. Cells are weakly or not fluorescent. × 300.
458
Fig. 3. Immunofluorescence of K5-5 cells after 4 days in culture, with an anti-SV-40 T serum. After fixation in acetone methanol (7/3, 10 min, --20 °C), cells were incubated successively with an Hamster anti-SV-40 tumor serum (1/10) (a gift of Dr. V. Defendi) and a fluorescein-conjugated rabbit antihamster immunoglobulin. Each incubation was done at 37 °C for 45 rain. Only nuclei are brightly fluorescent. × 300. A permanent clone of astroglial cells has been obtained from 14-day post-natal mice cerebella after transformation 'in vitro' with a wild type SV-40. At this stage o f cerebellar development, none o f the cells which incorporate [3H]thymidine mature into astrocytes (A. Privat, personal communication). It is possible that precursor cells for astrocytes are still present in these cerebella and that they start dividing when the tissue is dissociated and the cells put into culture. Furthermore, SV-40 may induce, or promote, the multiplication of these cells. Clone.1 cell lines with differentiated properties have been obtained after SV-40 transformation o f cultures from hypothalamus 5, chorion z or liver 9. The last two cell lines were transformed with a ts-A m u t a n t of SV40 and differentiated essentially at the non-permissive temperature at which cells were not transformed. Although the cerebellum astroglial cells we describe in this paper were obtained from cultures infected with a wild type SV-40, they show clear signs of transformation (presence o f SV-40 T-antigen and high rate o f cell multiplication), yet
459 synthesize GFA. These cells may be useful for studies on the interactions of astroglial cells with other cells of the CNS, such as neurones isolated from cerebeUum4, and also for the production of astroglial specific monoclonal antibodies. We are grateful to Dr. A. Bignami for the anti-GFA sera, to Dr. V. Defendi for the SV-40 and the anti-SV-40 T serum, and to Dr. C. Jaque for human GFA. This work was supported by C.N.R.S., I.N.S.E.R.M. and D.G.R.S.T. Grant 787-2777.
1 Bignami, A. and Stoolmiller, A. C., Astroglia-specific protein (GFA) in clonal cell lines derived from the G26 mouse glioma, Brain Research, 163 (1979) 353-357. 2 Bissell, M. G., Rubinstein, L. J., Bignami, A. and Herman, M. M., Characteristics of the rat C-6 glioma maintained in organ culture systems. Production of glial fibrillary acidic protein in the absence of gliofibriliogenesis, Brain Research, 82 (1974) 77-89. 3 Chou, J. Y., Establishment of clonal human placental cells synthesizing human choriogonadotropin, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1854--1858. 4 Cohen, J., Balazs, R., Hajos, F., Currie, D. N. and Dutton, R., Separation of cell types from the developing cerebellum, Brain Research, 148 (1978) 313-331. 5 De Vitry, F., Camier, M., Czernichow, P., Benda, Ph., Cohen, P. and Tixier-Vidal, A., Establishment of a clone of mouse hypothalamic neurosecretory cells synthesizing neurophysin and vasopressin, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 3575-3579. 6 Pessac, B., Alliot, F., Girard, A., Combes, P., Gu6rinot, F., Privat, A. and Drian, M. J., Properties of mouse cerebellum cells transformed by SV-40, Neurosci. Lett., Suppl. 1 (1978) $39. 7 Pope, J. H. and Rowe, W. P., Detection of specific antigen in SV-40-transformed cells by immunofluorescence, J. exp. Med., 120 (1964) 121-128. 8 Raju, T. R., Bignami, A. and Dahl, D., Glial fibrillary acidic protein in monolayer cultures of C-6 glioma cells: effect of aging and dibutyryl cyclic AMP, Brain Research, 200 (1980) 225-230. 9 Schlegel-Haueter, S. E., Schlegel, W. and Chou, J. Y., Establishment of a fetal rat liver cell line that retains differentiated liver functions, Proc. nat. Acad. Sci. (Wash.), 77 (1980) 2731-2734. 10 Stavrou, D., Rieske, E., Anzil, A. P., Haglid, K. G. and Isenberg, G., Definition of a cell clone with astroglial characteristics derived from a chemically induced rabbit brain glioma, J. neuroL Sci., 45 (1980) 287-301.
455
A glial fibrillary acidic protein (GFA)-containing cell clone derived from mouse cerebella transformed 'in vitro' by SV-40
F. ALLIOT and B. PESSAC* C.N.R.S. E.R. 231 and LN.S.E.R.M. U178, H@ital Broussais, 96 rue Didot, 75674 Paris Cedex 14 (France)
(Accepted March 5th, 1981) Key words: mouse - - cerebellum - - SV-40 - - cell clone - - glial fibrillary acidic protein - - T-antigen
A clonal cell line containing the glial fibrillary acidic protein (GFA) was derived from cultures of 14 day post-natal C57B1 mouse cerebella, transformed by SV-40. All the cells are GFA-positive - even in sparse exponential cultures - - have an astroglial morphology and contain the intranuclear SV40 T-antigen. The glial fibrillary acidic protein (GFA)-containing cell clones which have been reported in the literature were obtained from cultures isolated from chemically induced brain tumorsl,~, 10. This approach suffers from the drawback of not being able to determine the tissue of origin of these tumors. In an attempt to obtain cell lines from the various cell types of cerebellum, we transformed, with SV-40, cultures of mouse cerebellum cells taken at various stages of embryonic and postnatal development 6. Several cultures and clones have been established permanently in this way; in the present paper we confine our description to a GFA-positive clone derived from 14-day post-natal C57B1 mouse cerebella. Mice were sacrificed by decapitation, and cerebella quickly removed. The meninges were dissected out and the cerebella cut into small pieces. The tissue was dissociated by gentle pipetting in Eagle's basal medium (BME). Single cells and clumps were plated in tissue culture dishes coated with poly-L-lysine (10/~g/ml) in B M E q15 % fetal bovine serum (FBS) and immediately infected with about 10 FFU/cell of a wild type SV-40 (LP strain Rh 911, a gift of Dr. V. Defendi). The medium was renewed the following day and every week thereafter. One week after infection, the dishes were covered with confluent cell monolayers. Foci of apparently proliferating cells appeared throughout the culture after 25-30 days. The first subculture was done two months later, at a ratio of 1/3, and cloning started at the third subculture. The cultures were cloned by dilution because initial attempts to clone single isolated cells were unsuccessful. Single cells and small clumps (2-5 cells) dissociated from the culture by pipetting *To whom correspondence should be addressed.
456
Fig. 1. A and B: immunofluorescence, with 1/40 GFA antiserum, of the mouse cerebellum clonal cells K5-5. A, after 4 days and B, after 7 days in culture. The cytoplasm and processes are brightly fluorescent. x 300.
in BME + 20 ~ FBS, were mixed with 5 x 104 cells/ml of the same lethally irradiated culture which served as a feeder. The cell suspension was plated on Costar 'Tissue culture cluster 96' according to the Poisson distribution. This procedure was repeated 5 times over a 2.5 year period. Cell morphology remained heterogeneous, suggesting that the cultures were made up of two or more cell types. Only then were we able to obtain single cell clones by plating very diluted suspensions on coverslip fragments. One of the clonal cell lines tested, named CBl4-SV-4A4K5-5 (referred to in brief as K5-5), contained G F A (detected by indirect immunofluorescence with anti-GFA sera) and could therefore be identified as astroglial. The assay was performed on cells which were grown for 4-8 days on glass coverslips in tissue culture flasks, in BME t 10/o°/FBS in 95 ~ air, 5 ~ CO2, washed 3 times in PBS and fixed by immersion in acetone at - - 2 0 °C for 5 rain. The cells were then successively incubated in normal goat serum (l/6, l0 min), rabbit anti-GFA serum (batch numbers 35 and 75, a gift of Dr. A. Bignami) at a dilution of 1/40 for 120 min, and fluorescein-conjugated goat anti-rabbit immunoglobulin (Nordic Immunological Laboratories, Tilburg, Netherlands; 1/20 for 30 min). All incubations were done at room temperature and were followed by 3 rinses in PBS. The coverslips were mounted on slides in buffered glycerol and viewed with a Leitz epifluorescence microscope with appropriate filters.
457 In sparse cultures, all cells were brightly immunofluorescent and staining was confined to cytoplasm and processes (Fig. 1A, B). Their morphology was reminiscent of that of the other astroglial clones 1,2,10, with a thin rim of cytoplasm around a large nucleus and with few processes. It did not change in the 6 months following the single cell cloning and has remained constant ever since. All the cells were GFA-positive in both the exponential and stationary growth phases but extended more processes in the stationary phase. Although the specificity of these anti-GFA sera, as a marker for astroglial cells in culture, has been established 8, non-immune sera and immune sera not reacting to G F A (like an anti-tetanus toxoid serum), were used as controls, and were negative. Further evidence for the specificity of the anti-GFA sera came from the fact that staining was abolished by preincubation with human G F A (a gift of Dr. C. Jaque) (Fig. 2). The intranuclear SV-40 T antigen 7 was present in all ceils, thus proving that they were actually transformed by SV-40 (Fig. 3). The cells of clone K5-5 grow readily in BME -k 1 0 ~ FBS with a doubling time of about 24 h.
Fig. 2. Immunofluorescenceof K5-5 cells after 4 days in culture, with a 1/40 GFA antiserum absorbed with human GFA. Cells are weakly or not fluorescent. × 300.
458
Fig. 3. Immunofluorescence of K5-5 cells after 4 days in culture, with an anti-SV-40 T serum. After fixation in acetone methanol (7/3, 10 min, --20 °C), cells were incubated successively with an Hamster anti-SV-40 tumor serum (1/10) (a gift of Dr. V. Defendi) and a fluorescein-conjugated rabbit antihamster immunoglobulin. Each incubation was done at 37 °C for 45 rain. Only nuclei are brightly fluorescent. × 300. A permanent clone of astroglial cells has been obtained from 14-day post-natal mice cerebella after transformation 'in vitro' with a wild type SV-40. At this stage o f cerebellar development, none o f the cells which incorporate [3H]thymidine mature into astrocytes (A. Privat, personal communication). It is possible that precursor cells for astrocytes are still present in these cerebella and that they start dividing when the tissue is dissociated and the cells put into culture. Furthermore, SV-40 may induce, or promote, the multiplication of these cells. Clone.1 cell lines with differentiated properties have been obtained after SV-40 transformation o f cultures from hypothalamus 5, chorion z or liver 9. The last two cell lines were transformed with a ts-A m u t a n t of SV40 and differentiated essentially at the non-permissive temperature at which cells were not transformed. Although the cerebellum astroglial cells we describe in this paper were obtained from cultures infected with a wild type SV-40, they show clear signs of transformation (presence o f SV-40 T-antigen and high rate o f cell multiplication), yet
459 synthesize GFA. These cells may be useful for studies on the interactions of astroglial cells with other cells of the CNS, such as neurones isolated from cerebeUum4, and also for the production of astroglial specific monoclonal antibodies. We are grateful to Dr. A. Bignami for the anti-GFA sera, to Dr. V. Defendi for the SV-40 and the anti-SV-40 T serum, and to Dr. C. Jaque for human GFA. This work was supported by C.N.R.S., I.N.S.E.R.M. and D.G.R.S.T. Grant 787-2777.
1 Bignami, A. and Stoolmiller, A. C., Astroglia-specific protein (GFA) in clonal cell lines derived from the G26 mouse glioma, Brain Research, 163 (1979) 353-357. 2 Bissell, M. G., Rubinstein, L. J., Bignami, A. and Herman, M. M., Characteristics of the rat C-6 glioma maintained in organ culture systems. Production of glial fibrillary acidic protein in the absence of gliofibriliogenesis, Brain Research, 82 (1974) 77-89. 3 Chou, J. Y., Establishment of clonal human placental cells synthesizing human choriogonadotropin, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1854--1858. 4 Cohen, J., Balazs, R., Hajos, F., Currie, D. N. and Dutton, R., Separation of cell types from the developing cerebellum, Brain Research, 148 (1978) 313-331. 5 De Vitry, F., Camier, M., Czernichow, P., Benda, Ph., Cohen, P. and Tixier-Vidal, A., Establishment of a clone of mouse hypothalamic neurosecretory cells synthesizing neurophysin and vasopressin, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 3575-3579. 6 Pessac, B., Alliot, F., Girard, A., Combes, P., Gu6rinot, F., Privat, A. and Drian, M. J., Properties of mouse cerebellum cells transformed by SV-40, Neurosci. Lett., Suppl. 1 (1978) $39. 7 Pope, J. H. and Rowe, W. P., Detection of specific antigen in SV-40-transformed cells by immunofluorescence, J. exp. Med., 120 (1964) 121-128. 8 Raju, T. R., Bignami, A. and Dahl, D., Glial fibrillary acidic protein in monolayer cultures of C-6 glioma cells: effect of aging and dibutyryl cyclic AMP, Brain Research, 200 (1980) 225-230. 9 Schlegel-Haueter, S. E., Schlegel, W. and Chou, J. Y., Establishment of a fetal rat liver cell line that retains differentiated liver functions, Proc. nat. Acad. Sci. (Wash.), 77 (1980) 2731-2734. 10 Stavrou, D., Rieske, E., Anzil, A. P., Haglid, K. G. and Isenberg, G., Definition of a cell clone with astroglial characteristics derived from a chemically induced rabbit brain glioma, J. neuroL Sci., 45 (1980) 287-301.