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The inducibility of glycerol phosphate dehydrogenase in two rat glial clones
Brain Research, 109 (1976) 423-425
423
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
The inducibility of glycerol phosphate dehydrogenase in two rat glial clones
P. J. CLAISSE ANDJ. P. ROSCOE The School of Pathology, The Middlesex Hospital Medical School, London WIP 7LD (Great Britain)
(Accepted March 1st, 1976)
Although cytoplasmic glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) is found in several tissues, its inducibility by hydrocortisone (HC) is reported to be confined to neural tissueS, 10. This inducibility has been demonstrated in the following; the rat brain in vivo 9-1a, brain explant culture 5, dissociated rat cerebral cell culture 6, reaggregating brain cell cultures 7 and the cloned astrocytoma line C68,10, 14,15. This response is not detectable in fibroblasts s,14, ependymal, meningeal or neuronal cells 14 in vitro and is greatly reduced in hybrids between glial cells and fibroblasts s. It appears that the inducibility of this enzyme is a specialised function of glia. However, to our knowledge C6 (ref. 2) is the only cloned glial cell line in which the elevation of G P D H activity has been reported so far. In this study we sought to extend the correlation of inducibility with cell-type, by investigating the response of two glial clones and a number of other cultures to the hormone. A15A5 and A15A10 are two cell lines cloned in agar from the culture of a mixed glioma, 1A2, which was transplacentally induced by ethylnitrosourea in a BDIX rat 16,19. Both clones formed processes extensively when exposed to dibutyryl cyclic AMP (dbcAMP) z0 and gave rise to tumours histologically identified as malignant astrocytomas when injected into syngeneic hosts. The cells possessed the ultrastructural characteristics of fibrillary astrocytes 16. The cultures tested for G P D H inducibility by HC were: A15A5, A15A10, two cultures of the rat glial clone C6 (I and II), the mouse neuroblastoma C13001, an embryo rat fibroblast culture RE 4 and a non-tumour clone of adult rat brain fibroblasts ARBOC9. C6I and C1300 were gifts from Dr. G. Sato, C61I and RE from Dr. K. Fields. RE was derived by Dr. R. Bomford at the Imperial Cancer Research Fund, Lincoln's Inn Fields and ARBOC9 was prepared from a BDIX rat and cloned in this laboratory by Dr. C. J. Skidmore. Glass roller bottles were inoculated with 5 × 106 cells in 200 ml of growth medium (Dulbecco's modification of Eagle's medium with 15 ~o foetal calf serum), gassed with a 15 ~o CO~85 ~o air mixture and incubated at 37 °C. At the end of the log phase of growth, the period at which de Vellis found G P D H inducibility to be maximum in some C6 cultures 10, half the bottles received 2.6/~g/ml of hydrocortisone21-sodium succinate. After a further 48 h incubation the cells were collected by
424 TABLE -l THE
EFFECTS
OF H Y D R O C O R T I S O N E
ON
THE
ACTIVITY
OF GLYCEROL
PHOSPHATE
DEHYDROGENASI
IN
VARIOUS CULTURES
Hydrocortisone succinate was added at a final concentration of 2.6 F~g/ml medium.
Cell line
Control S.A. *
Plus hydrocortisone S.A.
Plus hydrocortisone ( % control)
C6I C611 A15A5 AI5AI0 C1300 RE ARBOC9
60.3 39.3 7.0 7.0 22.0 3.0 5.0
105.0 236.0 17.0 13.0 22.0 3.0 5.0
166.70 605.13 242.85 185.71 100.00 100.00 100.00
* Enzyme activity was measured 48 h later and is expressed as change in absorbance units/rag/rain × 10 -3 (S.A.).
trypsinization, rinsed with 0.1 M phosphate buffer, pH 7.3, and broken by homogenization. The extracts were centrifuged for 1 h at 100,000 × g and the supernatants tested for GPDH activity. Protein was determined by the method of Lowry et al. 17. GPDH activity was determined, within 3 h of homogenization, by the method of de Vellis et al. specific for cytoplasmic GPDH 13. The reaction mixture contained I mM dihydroxyacetone phosphate, 0.166 mM NADH, 6.6 mM nicotinamide and 1 mM amytal in 0.1 M phosphate buffer pH 7.3. Specific activity is expressed as change in absorbance units per mg protein per min. The results are presented in Table I. The glial clones AI5A5 and AI5A10 and the two C6 cultures showed marked inducibility of GPDH by HC. This distinguishes them from the other cultures tested. Data for C1300, RE and ARBOC9 confirm that fibroblast and neuronal cells in culture are not inducible. Our results reveal a high baseline in C1300 and low baselines in the whole embryo fibroblast culture and the fibroblast clone derived from adult brain. Our C6 cultures have widely differing basal activities, echoing a report by de Vellis who found a broad baseline spectrum in the C6 cultures and subclones he tested 1°. The comparatively low basal levels of A 15A5 and A 15AI0 could be regarded as representing one extreme of this wide range. The activity of GPDH was not induced by dbcAMP in A15A5. Similarly de Vellis reported that C6 did not respond to dbcAMP, a result consistent with the fact that HC does not cause cyclic AMP levels to rise in these cellsl°,lL Cells in the C6 cultures tested by de Vellis had karyotypes indistinguishable from that of normal rat cells1°. A 15A5 and A 15A 10 also have a modal number of 42 chromosomes, Our preliminary results suggest that GPDH may not be inducible in cells with abnormal karyotypes. The difficulty of identifying glia in culture is accentuated by the paucity of biochemical features which can be attributed uniquely to these cells. The protein S100 ~s and the fibrillary acidic protein, which is specific for the fibrillary astrocytesa,
425 2z are considered to be in this category. However, the specificity of the former has recently been questioned 21. The inducibility o f G P D H by H C should therefore be a valuable m a r k e r in establishing the identity o f putative glial lines, especially when considered in c o n j u n c t i o n with s u p p o r t i n g investigations. This study was supported in p a r t by a g r a n t f r o m the Cancer Research Camp a i g n to J. P. Roscoe. 1 AUGUSTbTocco, G., AND SATO, G., Establishment of functional clonal lines of neurons from mouse neuroblastoma, Proc. nat. A cad. Sci. (Wash.), 64 (1969) 311-315. 2 BENDA,P., LIGHTBODY,J., SATO,G., LEVINE,L., AND SWEET,W., Differentiated rat glial cell strain in tissue culture, Science, 161 (1968) 370-371. 3 BIGNAMI,A., ENG, L. F., DAHL,D., AND UYEDA,C. T., Localisation of the ra glial fibrillary acidic protein in astrocytes by immunofiuorescence, Brain Research, 43 (1972) 429-435. 4 BOrea~ORD,R., AND WEINSTEIN,Io B., Transformation of a rat epithelial-like cell line by murine sarcoma virus, J. nat. Cancer Inst., 49 (1972) 379-386. 5 BREEN,G. A. M., AND DE VELIS,J., Regulation of glycerol phosphate dehydrogenase by h~drocortisone in rat brain explants, Exp. Cell Res., 91 (1975) 159-169. 6 BREEN,G. A. M., ANDDE VELLIS,J., Regulation of glycerol phosphate dehydrogenase by hydrocortisone in dissociated rat cerebral cell cultures, Develop. Biol., 41 (1974) 255-266. 7 BREEN,G. A. M., DE VELLIS,J., AND COLE, R., In Trans. Soc. Neurosci, 3rd Annual Meeting, San Diego, 1973, p. 427. 8 DAVIDSON,R. L., AND BENDA, P., Regulation of specific functions of glial cells in somatic hybrids II. Control of inducibility of glyceroi-3-phosphate dehydrogenase, Proc. nat. Acad. Sci. (Wash.), 67 (1970) 1870-1877. 9 De VELLIS,J., Glycolysis in rat brain tissue slices following neonatal head x-irradiation: relation of regional differences to the LDH:GPDH ratio, J. Neurochem., 15 (1968) 1057-1060. 10 DE VELLIS,J., Mechanisms of enzymatic differentiation in the brain and in cultured cells. In M. ROCKSTEIN(Ed.), Development and Ageing in the Nervous System, Academic Press, New York, 1973, pp. 171-193. 11 DE VELLm,J., ANDINGLISH,D., Hormonal control of glycerolphosphate dehydrogenase in the rat brain, J. Neurochem., 15 (1968) 1061-1070. 12 DE VELLIS,J., AND SCHJEIDE,O. A., Time dependence of the effect of x-irradiation on the formation of glycerolphosphate dehydrogenase on the developing rat brain, Biochem. J., 107 (1968) 259-264. 13 DE VELLIS,J., SCHJEIDE,O. A., ANDCLEMENTE,C. D., Protein synthesis and enzymic patterns in the developing rat brain following x-irradiation of new born rats, J. Neurochem., 14 (1967) 499-511. 14 DE VELLtS,J., INGLISH,D., ANDGALEY,F., Effects ofcortisol and epinephrineon glial cells in culture. In D. PEASE(Ed.), Cellular Aspects of Growth and Differentiation in Nervous Tissue, Univ. Calif. Press, Berkeley, 1971, pp. 23-32. 15 DE VELLIS,J., INGLISH,D., COLE,R., ANDMOLSON,J., Effects of hormones on the differentiation of cloned lines of neurons and glial cells. In D. FORD (Ed.), Influence of Hormoneson the Nervous System, Karger, Basel, 1971, pp. 25-39. 16 LANTOS,P. L., ROSCOE,J. P., AND SKIDMORE,C. J., Studies of the morphology and tumorigenicity of experimental brain tumours in tissue culture, Brit. J. exp. Path., in press. 17 LOWRY,O. H., ROSEBROUGH,N. J., FARR,A. L., ANDRANDALL,R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 18 PFEIFFER,S. E., Clonal lines of glial cells. In G. SATO(Ed.), Tissue Culture of the Nervous System, Plenum, New York, 1973, pp. 203-230. 19 RoscoE, J. P., ANDGIBaS,B. E., Several changes associated with the acquisition of a single chromosome in rat glial tumour cells, J. nat. Cancer Inst., 53 (1974) 581-583. 20 ROSCOE,J. P., LANTOS,P. L., DAVIES,S., AND PILKINGTON,G. J., unpublished observations. 21 SCHUBERT,n., HEINEMANN,S., CARLISLE,W., TARIKAS,H., KIMES,B., PATRICK,J., STEINBACH,J. H., CULP, W., AND BRANDT,B. L., Clonal lines from the rat central nervous system, Nature (Lond.), 249 (1974) 224-227. 22 UYEDA, C. T., ENG, L. F., AND BIGNAMI, k., Immunological study of the glial fibrillary acidic protein, Brain Research, 37 (1972) 81-89.
423
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
The inducibility of glycerol phosphate dehydrogenase in two rat glial clones
P. J. CLAISSE ANDJ. P. ROSCOE The School of Pathology, The Middlesex Hospital Medical School, London WIP 7LD (Great Britain)
(Accepted March 1st, 1976)
Although cytoplasmic glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) is found in several tissues, its inducibility by hydrocortisone (HC) is reported to be confined to neural tissueS, 10. This inducibility has been demonstrated in the following; the rat brain in vivo 9-1a, brain explant culture 5, dissociated rat cerebral cell culture 6, reaggregating brain cell cultures 7 and the cloned astrocytoma line C68,10, 14,15. This response is not detectable in fibroblasts s,14, ependymal, meningeal or neuronal cells 14 in vitro and is greatly reduced in hybrids between glial cells and fibroblasts s. It appears that the inducibility of this enzyme is a specialised function of glia. However, to our knowledge C6 (ref. 2) is the only cloned glial cell line in which the elevation of G P D H activity has been reported so far. In this study we sought to extend the correlation of inducibility with cell-type, by investigating the response of two glial clones and a number of other cultures to the hormone. A15A5 and A15A10 are two cell lines cloned in agar from the culture of a mixed glioma, 1A2, which was transplacentally induced by ethylnitrosourea in a BDIX rat 16,19. Both clones formed processes extensively when exposed to dibutyryl cyclic AMP (dbcAMP) z0 and gave rise to tumours histologically identified as malignant astrocytomas when injected into syngeneic hosts. The cells possessed the ultrastructural characteristics of fibrillary astrocytes 16. The cultures tested for G P D H inducibility by HC were: A15A5, A15A10, two cultures of the rat glial clone C6 (I and II), the mouse neuroblastoma C13001, an embryo rat fibroblast culture RE 4 and a non-tumour clone of adult rat brain fibroblasts ARBOC9. C6I and C1300 were gifts from Dr. G. Sato, C61I and RE from Dr. K. Fields. RE was derived by Dr. R. Bomford at the Imperial Cancer Research Fund, Lincoln's Inn Fields and ARBOC9 was prepared from a BDIX rat and cloned in this laboratory by Dr. C. J. Skidmore. Glass roller bottles were inoculated with 5 × 106 cells in 200 ml of growth medium (Dulbecco's modification of Eagle's medium with 15 ~o foetal calf serum), gassed with a 15 ~o CO~85 ~o air mixture and incubated at 37 °C. At the end of the log phase of growth, the period at which de Vellis found G P D H inducibility to be maximum in some C6 cultures 10, half the bottles received 2.6/~g/ml of hydrocortisone21-sodium succinate. After a further 48 h incubation the cells were collected by
424 TABLE -l THE
EFFECTS
OF H Y D R O C O R T I S O N E
ON
THE
ACTIVITY
OF GLYCEROL
PHOSPHATE
DEHYDROGENASI
IN
VARIOUS CULTURES
Hydrocortisone succinate was added at a final concentration of 2.6 F~g/ml medium.
Cell line
Control S.A. *
Plus hydrocortisone S.A.
Plus hydrocortisone ( % control)
C6I C611 A15A5 AI5AI0 C1300 RE ARBOC9
60.3 39.3 7.0 7.0 22.0 3.0 5.0
105.0 236.0 17.0 13.0 22.0 3.0 5.0
166.70 605.13 242.85 185.71 100.00 100.00 100.00
* Enzyme activity was measured 48 h later and is expressed as change in absorbance units/rag/rain × 10 -3 (S.A.).
trypsinization, rinsed with 0.1 M phosphate buffer, pH 7.3, and broken by homogenization. The extracts were centrifuged for 1 h at 100,000 × g and the supernatants tested for GPDH activity. Protein was determined by the method of Lowry et al. 17. GPDH activity was determined, within 3 h of homogenization, by the method of de Vellis et al. specific for cytoplasmic GPDH 13. The reaction mixture contained I mM dihydroxyacetone phosphate, 0.166 mM NADH, 6.6 mM nicotinamide and 1 mM amytal in 0.1 M phosphate buffer pH 7.3. Specific activity is expressed as change in absorbance units per mg protein per min. The results are presented in Table I. The glial clones AI5A5 and AI5A10 and the two C6 cultures showed marked inducibility of GPDH by HC. This distinguishes them from the other cultures tested. Data for C1300, RE and ARBOC9 confirm that fibroblast and neuronal cells in culture are not inducible. Our results reveal a high baseline in C1300 and low baselines in the whole embryo fibroblast culture and the fibroblast clone derived from adult brain. Our C6 cultures have widely differing basal activities, echoing a report by de Vellis who found a broad baseline spectrum in the C6 cultures and subclones he tested 1°. The comparatively low basal levels of A 15A5 and A 15AI0 could be regarded as representing one extreme of this wide range. The activity of GPDH was not induced by dbcAMP in A15A5. Similarly de Vellis reported that C6 did not respond to dbcAMP, a result consistent with the fact that HC does not cause cyclic AMP levels to rise in these cellsl°,lL Cells in the C6 cultures tested by de Vellis had karyotypes indistinguishable from that of normal rat cells1°. A 15A5 and A 15A 10 also have a modal number of 42 chromosomes, Our preliminary results suggest that GPDH may not be inducible in cells with abnormal karyotypes. The difficulty of identifying glia in culture is accentuated by the paucity of biochemical features which can be attributed uniquely to these cells. The protein S100 ~s and the fibrillary acidic protein, which is specific for the fibrillary astrocytesa,
425 2z are considered to be in this category. However, the specificity of the former has recently been questioned 21. The inducibility o f G P D H by H C should therefore be a valuable m a r k e r in establishing the identity o f putative glial lines, especially when considered in c o n j u n c t i o n with s u p p o r t i n g investigations. This study was supported in p a r t by a g r a n t f r o m the Cancer Research Camp a i g n to J. P. Roscoe. 1 AUGUSTbTocco, G., AND SATO, G., Establishment of functional clonal lines of neurons from mouse neuroblastoma, Proc. nat. A cad. Sci. (Wash.), 64 (1969) 311-315. 2 BENDA,P., LIGHTBODY,J., SATO,G., LEVINE,L., AND SWEET,W., Differentiated rat glial cell strain in tissue culture, Science, 161 (1968) 370-371. 3 BIGNAMI,A., ENG, L. F., DAHL,D., AND UYEDA,C. T., Localisation of the ra glial fibrillary acidic protein in astrocytes by immunofiuorescence, Brain Research, 43 (1972) 429-435. 4 BOrea~ORD,R., AND WEINSTEIN,Io B., Transformation of a rat epithelial-like cell line by murine sarcoma virus, J. nat. Cancer Inst., 49 (1972) 379-386. 5 BREEN,G. A. M., AND DE VELIS,J., Regulation of glycerol phosphate dehydrogenase by h~drocortisone in rat brain explants, Exp. Cell Res., 91 (1975) 159-169. 6 BREEN,G. A. M., ANDDE VELLIS,J., Regulation of glycerol phosphate dehydrogenase by hydrocortisone in dissociated rat cerebral cell cultures, Develop. Biol., 41 (1974) 255-266. 7 BREEN,G. A. M., DE VELLIS,J., AND COLE, R., In Trans. Soc. Neurosci, 3rd Annual Meeting, San Diego, 1973, p. 427. 8 DAVIDSON,R. L., AND BENDA, P., Regulation of specific functions of glial cells in somatic hybrids II. Control of inducibility of glyceroi-3-phosphate dehydrogenase, Proc. nat. Acad. Sci. (Wash.), 67 (1970) 1870-1877. 9 De VELLIS,J., Glycolysis in rat brain tissue slices following neonatal head x-irradiation: relation of regional differences to the LDH:GPDH ratio, J. Neurochem., 15 (1968) 1057-1060. 10 DE VELLIS,J., Mechanisms of enzymatic differentiation in the brain and in cultured cells. In M. ROCKSTEIN(Ed.), Development and Ageing in the Nervous System, Academic Press, New York, 1973, pp. 171-193. 11 DE VELLm,J., ANDINGLISH,D., Hormonal control of glycerolphosphate dehydrogenase in the rat brain, J. Neurochem., 15 (1968) 1061-1070. 12 DE VELLIS,J., AND SCHJEIDE,O. A., Time dependence of the effect of x-irradiation on the formation of glycerolphosphate dehydrogenase on the developing rat brain, Biochem. J., 107 (1968) 259-264. 13 DE VELLIS,J., SCHJEIDE,O. A., ANDCLEMENTE,C. D., Protein synthesis and enzymic patterns in the developing rat brain following x-irradiation of new born rats, J. Neurochem., 14 (1967) 499-511. 14 DE VELLtS,J., INGLISH,D., ANDGALEY,F., Effects ofcortisol and epinephrineon glial cells in culture. In D. PEASE(Ed.), Cellular Aspects of Growth and Differentiation in Nervous Tissue, Univ. Calif. Press, Berkeley, 1971, pp. 23-32. 15 DE VELLIS,J., INGLISH,D., COLE,R., ANDMOLSON,J., Effects of hormones on the differentiation of cloned lines of neurons and glial cells. In D. FORD (Ed.), Influence of Hormoneson the Nervous System, Karger, Basel, 1971, pp. 25-39. 16 LANTOS,P. L., ROSCOE,J. P., AND SKIDMORE,C. J., Studies of the morphology and tumorigenicity of experimental brain tumours in tissue culture, Brit. J. exp. Path., in press. 17 LOWRY,O. H., ROSEBROUGH,N. J., FARR,A. L., ANDRANDALL,R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 18 PFEIFFER,S. E., Clonal lines of glial cells. In G. SATO(Ed.), Tissue Culture of the Nervous System, Plenum, New York, 1973, pp. 203-230. 19 RoscoE, J. P., ANDGIBaS,B. E., Several changes associated with the acquisition of a single chromosome in rat glial tumour cells, J. nat. Cancer Inst., 53 (1974) 581-583. 20 ROSCOE,J. P., LANTOS,P. L., DAVIES,S., AND PILKINGTON,G. J., unpublished observations. 21 SCHUBERT,n., HEINEMANN,S., CARLISLE,W., TARIKAS,H., KIMES,B., PATRICK,J., STEINBACH,J. H., CULP, W., AND BRANDT,B. L., Clonal lines from the rat central nervous system, Nature (Lond.), 249 (1974) 224-227. 22 UYEDA, C. T., ENG, L. F., AND BIGNAMI, k., Immunological study of the glial fibrillary acidic protein, Brain Research, 37 (1972) 81-89.