Metabolic Brain Disease, Vol. 6, No. 1, 1991
Differential Regulation of Myelin Gene Expression in SV40 T Antigen-transfected Rat Glioma C6 Cells M. Bong, 1 A. Chakrabarti, 1 N. Banik, ~E.L. H o g a n , I M. K a n o h , 2 R . C . W i g g i n s , z and
G. KonaV :~
Received October 18, 1990; accepted January 16, 1991 Rat glioma C6 cells were stably Iransfected with a pSV3-neo plasmid containing SV40 T antigen gene, and geniticin-resistant transfectants (designated C6T ceils) were cloned. The C6T cells grew as well-defined foci of cells showing squamous or irregular morphology. The doubling time for transfected ceils was reduced by approximately 40% as compared to control C6 ceils. The transfecfion with T-antigen also affected the expression of genes coding for structural myelin proteins and for myelin-associated enzymes. The steady-state level of proteolipid protein (PLP)-speeific mRNA in C6T cells was 44% lower than in parental C6 cells. On the other hand, the transfection upregulated the expression of myelin-associated glycoprotein (MAG) by 153%. The activity of 2':3'cyclic AMP phosphodiesterase (CNP) was increased by approximately 80 % in the C6T cells as compared to untransfected, control ceils. The activity of calcium-activated neutral proteinase (CANP) was also significantly elevated in the transfectants by approximately 50% and 220% for millimolar and micromolar form respectively. The results indicate that T antigen affects the expression of myelin genes, although, individual genes appear to be differenflyregulated implying the existence of several independent regulatory mechanisms. KEY WORDS: C6 ceils; transfection; SV40 T antigen; gene expression;
myelin proteins; myelin enzymes.
INTRODUCTION Although, many important advances have been made within the past several years in our understanding of structures and biological properties of major myelin proteins at the 1 Department of Neurology, Medical University of South Carolina, 171 Ashley Ave., Charleston, S.C. 29425 32Departmen~ of Anatomy, West Virginia University School of Medicine, Mqrgantpwn, WV 26506. To whom correspondence should b~ addressed at Department of Anatomy, west virginia University, School of Medicine, 4052 HSN, Morgantown, WV 26505.
7 0~85-7490/91/0300-0007506.50/0 9 1991 Plenum Publishing Corporation
8
Konat et al.
molecular biological level, little is known about the factors that regulate the expression of corresponding genes. Cultured myelinating oligodendroeytes do not proliferate and this severely limits their use for genetic and biochemical studies of the process. The rat glioma C6 cell line is closely related to the oligodendrocyte, and has served as a model for the in vitro investigation of glial properties (Seeds et al., 1970; Clark and Perkins, 1971; Pfeiffer, 1973; Bachrach, 1975; Bennett et al., 1977; McMorris et al., 1985; Van Eldik and Zimmer, 1987; Reggiani et al., 1987; Herschman et al., 1973; Wolfe et al., 1980). Although the C6 cells do not produce myelin-related structures, they express some of the myelin-specific proteins, i.e., 2':3'-cyclic nncleotide 3'-phosphohydrolase (CNP) (McMorris et al., 1985) and proteolipid protein (PLP)(Volpe et al., 1975; Milner et al., 1985). We have found that these cells also express calcium-activated neutral proteinase (CANP)(Banik et al., 1988), an enzyme which is specifically localized in brain myelin (Banik et al., 1987). Recent advances in cell and molecular biological methodology open new possibilities to examine the genetic regulatory mechanisms by incorporation of foreign genes which would perturb the normal expression of the host genes. The aim of the present study was to determine whether the expression of myelin-specific genes in the C6 cells can be experimentally manipulated. The experimental paradigm employed transfection of the C6 cells with Simian Virus 40 (SV40) T antigen gene, which is a potent effector of mammalian gene expression (Tjian 1981; Mitchell et al., 1987).
MATERIALS AND METHODS Cell Culture
The C6 glioma cells were obtained from Dr. Kenneth Leskawa (University of Louisville, KY) and grown in Ham's F-10 medium/Dulbecco modified Eagle's medium (1:1) (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (Biofluids, Inc., Rockville, MD) and 1% of antibiotic-antimycotic mixture (Gibco) in plastic flasks or dishes at 37~ under 96% air/4% C02. For measurement of growth rate, the cells were plated in 6-weU (35 mm) tissue culture plates (Costar, Cambridge, MA) at 16 x 105 cells/well. At different intervals the cells were trypsinized and counted in a hemacytometer. cDNA Probes and Vectors
Plasmid p27 containing full-length eDNA (3.2 kb) for rat PLP (Milner et al., 1985) and plasmid plB236 containing partial eDNA (1.5 kb) for rat MAG (Sutcliffe et al., 1983) were obtained from Dr. Robert Milner (Scripps Clinic, La Jolla, CA). Plasmid pSV3-neo (Okayama & Berg 1983), containing the SV40 T antigen gene and selectable neo gene (providing resistance to geneticin) was obtained from the American Type Culture Collection.
Differential Regulation of Myelin Gene Expression
DNA Transfection
106 cells grown for 24 h in 10 cm plastic dish were transfected with pSV3-neo plasmid by calcium phosphate technique using CellPhect Transfection kit (Pharmacia). The culture was subsequently incubated for 12 hours at 37~ and subjected to osmotic shock by incubation in isotonic HEPES buffer, pH 7.5 containing 15% glycerol for 3 minutes. The ceils were rinsed, incubated in standard culture medium (containing 10% fetal calf serum) for 48 hours, trypsinized, diluted 1:2 and replated on new dishes with fresh medium. 12-14 hours later geneticin sulfate (G418) (Gibco) was added to the medium to a final concentration of 0.5 mg/ml. The drug-containing medium was changed every 4 days. After 14-16 days of selection, individual colonies (geneticin-resistant clones) were picked and transferred to 24-well plates (Costar, Cambridge, MA). The transfected cells, designated C6T, were passaged at confluence, expanded into 75 cm2 flasks and used for further experiments. Mock-transfected (without DNA) cells did not yield any G418-resistant colonies. R N A Isolation
Cells were washed with PBS, scraped and collected in sterile eppendorf tubes, and cytoplasmic RNA was isolated by the procedure of White and Bancroft (1982). The poly (A) + RNA was separated out on oligo(dT)-cellulose column as described by Maniatis et al., (1982). Poly (A)§ RNA was stored in 70% ethanol at -20~ prior to use. The concentration of nucleic acids was estimated by absorbance at 260 nm. Northern and Dot Blot Analysis
PLP and MAG radioactive probes were generated as previously described (Konat et al., 1988). For northern analysis, total RNA (10-30 ~tg) was denatured with formaldehyde, electrophoresed on 1% agarose gel and subsequently transferred in 10X SSC to BIOTRANS nylon membrane (ICN Biochemicals, Cleveland, OH). The membrane was further processed and hybridized with labeled eDNA probes (1-5 x 106 cpm/ml) according to the manufacturer's protocol. The position of the specific messages was detected by autoradiography. For quantitation, the autoradiograms were scanned using a LKB 2202 Ultroscan laser densitometer and the relative amount of radioactivity was determined by measuring the integrated optical density. Dot blot analysis was performed as described by Davis et al.(1986). Serial dilutions were made in 15X SSC, starting with 10 ~g of total RNA or 1-5/I.tg of poly (A)§ RNA denatured with formaldehyde. The samples were applied to BIOTRANS nylon membrane using dot blot apparatus and further processed and quantitated as described for northern blots. Cell Subfractionation and Protein Determination
Cells were washed twice with saline and homogenized in 20 mM Tris-acetate buffer (pH 7.5) containing 1 mM EDTA, 2.5 mM 2-mercaptoethanol, 1 mM PMSF (phenyl methyl sulfonyl fluoride) and lmM NAN3. Following centrifugation of the homogenate at 110,000
10
Konatet al.
g for 60 min pellet and supematant (cytosol) fractions were collected. The protein content was determined by the method of Smith et al (1985).
Determination of Enzymatic Activities
Adenosine 2',-cyclic nucleotide 3'-phosphohydrolase (CNP) activity was assayed by the method described earlier (Kurihara and Tsukada, 1967: Banik and Davison, 1969). One unit [U] of the enzyme activity corresponds to one, l&rnoleof 2'-AMP formed per hour. Micromolar calcium-activated neutral protease (gM CANP) and its millimolar isoform (raM CANP) were separated from inhibitor and partially purified by DEAE-cellulose and ~34henyl sepharose chromatography (Murachi, 1984; Chakrabarti and Banik 1988). C-azocasein substrate was prepared by reductive alkylation of azocasein (Sigma Chemical Co., St. Louis, MO) with 14C-formaldehyde (American Radiolabeled Chemicals Inc., St. Louis, MO), and the enzyme activities were assayed as previously described (Banik et al., 1987). Samples containing EGTA (5 mM) served as controls. One unit of activity [lJ] was defined as the amount of enzyme which liberates 103 cpm of acid-soluble radioactivity per hour. RESULTS
The rat glioma C6 cells were stably transfected with a pSV3-neo construct containing the SV40 T antigen gene (Okayama & Berg 1983) and the geniticin-resistant transfectants (designated C6T cells) were cloned. The relatively high transfection frequency of about one transformant in 6 x 104 cells demonstrates that the C6 cells effectively take up recombinant DNA molecules from the calcium phosphate precipitate and stably express its genes. The transfected, C6T cells were more tightly packed together than normal C6 cells and tended to grow over each other forming foci (Fig. 1). Most of the C6T cells showed irregular or squamous morphology and tended to detach from the substratum. These cells grew to a slightly higher saturation densities in monolayer cultures than did parental C6 cells. The doubling time for C6T cells was 11.3 hr as compared to 18 hr for the C6 cells. The transfection also caused doubling in the number of dead cells as compared to the parental cell line. Both C6 and C6T cells expressed myelin-specific protein genes, viz. PLP and MAG as evaluated by steady-state level of specific mRNA messages determined by hybridization technique (Fig. 2). Two PLP transcripts of 3.2 and 1.6 kb were detected by Northern blot analysis, whereas MAG eDNA probe hybridized to single regions of 2.5 kb. The size-pattern of the messages was not affected by the transfecfion of C6 ceils with T antigen, although, pronounced differences in their steady-state level were observed. Thus, the level of PLP messages in the transfectants was only 56% of that of the parental (control) cells (Table I). On the other hand, the transfection elevated MAG-specific mRNAs to 253% of control. The transfection of the C6 cells with T antigen also upregulated the expression of myelin-associated enzymes, i.e. CNP and CANP (Table If). Both total (expressed per 106 cells) and specific (expressed per mg of protein) CNP activity was elevated in C6T cells to
Differential Regulation of Myelin Gene Expression
11
Fig. 1. The morphological changes in rat glioma C6 cells induced by stable transfection with SV40 T antigen. Control (C6) cells (upper panel) and transfeeted (C6T) cells (lower panel) were grown as described in Materials and Methods and phase-contrast photographed at 200X magnification.
12
Konat et aL
PLP
MAG
C
C
T
T
Ftg. 2. ~xpression ot myelin protein genes in C6 ceils and in C6 ceils stably transfected with SV40 T antigen (C6T cells). 25 txg of cytoplasmic RNA were applied per lane and northern blot a n a l y s i s was performed as described in Materials and Methods with eDNA probes for PLP (panel A) and MAG (panel
B).
Table L The Effect of T antigen on PLP and MAG mRNA in the C6 cells. Message PLP MAG The relative messages was northern blot and Methods). + S.D. from 7
% of control C6 Cells 56 + 13 253 + 81 amount of specific determined by dot and analysis (see Materials Values represent means experiments
Differential Regulation of Myelin Gene Expression
13
Table H. The Effect of T antigen on CNP, mM CANP and uM CANP activity in C6 Cells
Enz),me CNP IxMCANP mM CANP
Total acdvit~ (U/106 Cells) C6 cells C6T ceils 301.2 + 12.2 542.2 + 11.4a 79 +- 18 252 • 24 a 474 4- 96 711 + 14IF
Specific Activit~ (U/mg protein) C6 cells C6T cells 54.0 + 6.6 97.2 + 6.2 a 149 + 28 478 • 82a 915 + 142 1373 + 204b
U - units of activity (cf. Materials and Methods). Values from four experiments are expressed as mean + S.D. Significance levels by t-test: a p < 0.001 ; b p < 0.02; c p < 0.05
approximately 180% of the parental cells. The activity (both total and specific) of millimolar and micromolar CANP was increased in the transfectants to 150 and 320% of control value, respectively. Approximately 88% of CNP, 78% of mMCANP and 15% of I.tMCANP activity was associated with particulate fraction of the C6 cell homogenate and the subcellular distribution of these three enzymatic activities was not affected by the transfection (results not shown).
DISCUSSION
The rat glioma C6 cells have been shown to express some of the myelin-specific genes, i.e. CNP (McMorris et al., 1985), PLP (Volpe et al., 1975; Milner et al., 1985) and CANP (Banik et al., 1988). In this communication we show that the C6 cells also express MAG, a myelin-associated glycoprotein thought to play a regulatory role in myelinogenesis (Quarles 1984). The MAG-specific mRNAs from the C6 cells migrate as a single band corresponding to 2.4-2.5 kb. This is the size range of MAG messages found in the rat brain, where the primary transcripts of MAG gene undergo alternative splicing resulting in two messages of 2,389 and 2,474 bp (Lai et al., 1987). Thus, the processing of MAG transcripts in the C6 cells appears (at this resolution) to be the same as in the oligodendrocytes. The size of PLP messages in the C6 cells, observed in this study, are congruent with those reported previously (Milner et al., 1985) and resemble the pattern found in the rat brain. The large T antigen of Simian Virus 40 (SV40) is a multifunctional phosphoprotein, which shares the ability to both activate and repress gene transcription in mammalian cells (Tjian, 1981; Mitchell et al., 1987). Other functions contribute to its ability to promote immortalization as well as neoplastic transformation of mammalian cells (Colby & Shenk 1982; Petit et al., 1983; Chou & Martin 1975; Asselin & Bastin 1985 Lewis & Martin 1979). The molecular mechanisms of these functions are as yet undefined but may be related to the ability of T antigen to form a tight complex with nuclear phosphoprotein pS3 (Crawford et al., 1981), to bind to double-stranded DNAs (Clark et al., 1983), to its protein
14
Konat et al.
phosphorylating activity (Griffin et a/., 1979), and/or to the combination of the above properties. It has been recently demonstrated that myelin-specific genes are also susceptible to the effects of T antigen. Chert et al., (1987) found that the transformation of Schwann cells with SV40 virus upregulates the expression of MAG and CNP genes. On the other hand, Tennekoon et al., (1987) observed downregulation of the expression of CNP by T antigen introduced into the Schwalm cells by transfection. The discrepancy may be due to different experimental paradigms, i.e. viral transformation vs. transfection with recombinant T antigen gene. This study shows that the T antigen also effects myelin genes in the CNS-derived C6 cells. Thus, PLP expression was downregulated to approximately half of its message level in the parental ceils, while the expression of other genes was upregulated in the C6T cells as compared to the control C6 cells (Table I & II, Fig. 3). The message sizes of PLP and MAG were not altered by the transfection with T antigen. Thus, T antigen seems to exert its effect(s) in the C6 cells at the genomic level and not to affect the posttranscriptional processing of the primary transcripts. Although, the mechanisms of regulatory action of T antigen remain unknown, individual genes appear to be differently regulated (reciprocal regulation of expression of PLP versus other genes)(Fig. 3) implying the existence of several independent regulatory mechanisms reflecting different arrays of cis- and trans-acting elements for individual genes. The mechanism of expression of myelin genes, i.e. the relationship between transcription and translation is not fully understood in the C6 cells. The levels of PLP and MAG proteins
350 300 250
:-----..-^" , A A A A f
f3_~ 200 KMMMM
,
15o
o 100 50 0
~KXx~ 9 PLP ~ MAG ~ CNP ~]p~M CANP [ ] mM CANP
Fig. 3. T antigen-induced alteration in the expression of myelin genes in rat glioma C6 cells. The values are calculated from data shown ill Table 1 andTable 2 and are expressed as percent of parental (untransfeeted) cells.
Differential Regulation of Myelin Gene Expression
15
in both parental and transfected C6 cells was below the detection by immunoblot analysis (results not shown). At this point it is not known, whether the mRNAs increase is accompanied by increased synthesis of MAG or, whether PLP synthesis is blocked by T antigen. However, the cropping and dumping of the C6T cells may be explained by an increased expression of MAG on their surface, and thus provide a circumstantial evidence for the translation of the induced messages. Furthermore, the increase in the amount of CNP and CANP (as expressed per either cell or protein, Table II) may indicate that the messages were normally translated in this system. In conclusion, the demonstration that the C6 cells normally express MAG in addition to other major myelin proteins extends the applicability of the C6 cells as a model for oligodendrocytes. Furthermore, the expression of the myelin-specific genes can be experimentally modulated, and hence, these cells provide a useful model system to study the mechanisms of regulation of these genes.
ACKNOWLEDGMENTS The authors wish to thank Dr. Maria Trojanowska for helpful advice and discussion during the course of this work and Ms. Joan Kingsley for secretarial assistance. This work was supported by NIH grants NS-12044, NS-11066, and NS-21353 from the NINDS.
REFERENCES
Asselin C & Bastin M (1985) Sequences fi'om polyoma virus and simian virus 40 large T genes capable of immortalizing primary rat embryo fibroblasts. J Viral 56:958-968. Bachrach U (1975) Cyclic AMP-mediated induction of ornithine decarboxylase of glioma and neuroblastoma cells. Proc Natl Acad Sci USA 72:308?-3091. Banik NL, Chakrabarfi AK, Gantt G & Hogan EL (1987) Distribution of calcium-activated neutral proteinase activity in quaking mouse brain: A subcellular study. Brain Res 435:57-62. Banik N, Chakrabarti A, Konat G & Hogan EL (1988) Ca2+-activated neutral proteinase in C6 cells. Trans Am Soc Neurochem 19, 10% Banik NL & Davison AN (1969) Enzyme activity and composition of myelin and subcellular fractions in the developing rat brain. Biochem J 115:1051-1062.. Bennett K, McGinnis JF & DeVellis J (1977) Reversible inhibition of the hydroconisone induction of glycerol phosphate dehydrogenase by cytochalasin B in rat glial C6 cells. J Cell Physial 93:247-260. Chakraharti AK & Banik NL (1988) Purification of calcium-activated neutral proteinase (CANP) from purified myelin of bovine brain white matter. Neurochem Res 13:127-134. Chert GL, Halligan NLN, Lue NF & Chert WW (1987) Biosynthesis of myelin-associated proteins in Simian virus 40 (SV40)-transformed rat Schwann cell lines. Brain Res 414:35-48. Chou J & Martin R (1975) DNA infectivity and the induction of DNA synthesis with temperature sensitive mutants of simian virus 40. J Viro115:145-155. Clark R, Peden K, Pipas J, Nathans D & Tjian R (1983) Biochemical activities ofT antigen proteins encoded by simian virus 40A gene deletion mutants. Mol Cell Biol 3:220-228.
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Clark RB & Perkins JP (1971) Regulation of adenosine 3',5'-cyclic monophosphate concentration in cultured human astrocytoma cells by catecholamines and histamine. Proc Nail Acad Sci 68:2757-2760. Colby W & Shenk T (1982) Fragment of the simian virus 40 transforming gene facilitates transformation of rat embryo cells. Proc Nat Acad Sci USA 79:5189-5193. Crawford L, Pim D, Gurney E, Goodfeliow P & Taylor-PapadimitriouJ (1981) Detection of a common feature in several human tumor cell lines-a 53,000 dalton protein.ProcNat AcadSci USA 78:41-45. Davis LG, Dibner MD & Battey IF (1986) Basic methods in molecular biology. New York: Elsevier Science Publishing Co, Inc. pp 147-149. Griffin J, Spangler G & Livingstone D (1979) Protein kinase activity associated with simian virus 40 tumor antigen. Proc Nat Acad Sci USA 76:2610-2614. Hersehman HR, Grauling BP & Lerner MP (1973) Nervous System-specificproteins in cultured neural cells. In Sato G (ed.), Tissue Culture of the Nervous System. Plenum Press, New York, pp. 187-202. Konat G, Trojanowska M, Gantt G & Hogan EL (1988) Expression of myelin protein genes in quaking mouse brain. JNeurosci Res 20:19-22. Kurihara T. & Tsukada Y. (1967) The regional and subcellulax distribution of 2', 3'- cyclic nucleotide 3'-phosphohydrolase in the central nervous system. J Neurochem 14:1167-1174. Lai C, Brow MA, Nave K-A, Norohna AB, Quarles RH, Bloom FE, Milner RJ & Sutcliffe JG (1987) Two forms of 1B236/myelin-associated glycoprotein, a cell adhesion molecule for postnatal neural development, are produced by alternative splicing. Proc Natl Acad Sci USA 84:4337-4341. Lewis AM & Martin RG (1979) Oncogenicity of simian virus 40 deletion mutants that induce altered 17-kilodalton t-proteins. Proc Nat Acad Sci USA 76:4299-4302. Maniatis T, Ffitsch EF & Sambrook J (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor NY, Cold Spring Harbor Lab, pp. 190-198. McMorris FA, Smith TM, SprinkleTJ & Auszmann JM (1985) Induction of myelin components: cyclic AMP increases the synthesis rate of 2',3'- cyclic nucleotide 3'-phosphohydrolase in C6 glioma ceils. J Ne urochem.44:1242-1251. Milner RJ, ~ C, Nave K-A, Lenoir D, Ogata J & 8uteliffe JG (1985) Nucleotide sequences of two mRNAs for rat brain myelin proteolipid protein. Cell 42:931-939. Mitchell PJ, Wang C & Tjian R (1987) Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is inhibited by 8V40 T antigen. Cell 50:847-861. Murachi T. (1984) Calcium-dependent proteinases and specific inhibitors: Calpain and calpastatin. Biochem Soc Symp 49:149-167. Okayama H & Berg P (1983) A eDNA cloning vector that permits expression of eDNA inserts in mammalian cells. Mol Cell Biol 3:280-289. Petit C, Gardes M & Feunteun J (1983) Immortalization of rodent embryo fibroblasts by SV40 is maintained by the A gene. Virology 127:74-82. Pfeiffer SE (1973) Clonal lines of glial cells. In 8ato G, (ed.), Tissue Culture of the Nervous System. Plenum Press, New York, pp 203-230. Quarles RH (1984) Myelin-associated glycoprotein in development and disease. Dev Neurosci 6:285-303. Reggiani A, Carenzi A & Della Bella D (1978) Influence of opioids on -receptors down-regulation: Studies in cultured C6 glioma cells. Brain Res 423:254-260. Seeds NW, Gilman AG, Armano T & Nirenberg M (1970) Regulation of axon formation by clonal lines of a neural tumor. Proc Nail Acad Sci 66:160-167. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gastner FH, Provenzano MD, Fujimoto El(, Goeke NM, Olson BJ & Klenk DC (1985) Measurement of protein using bicinchoninic acid. Analytical Biochem. 150:76-85.
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17
Sutcliffe JG, Milner RJ, Shinnick TM & Bloom FE (1983) Identifying the protein product of brain specific genes with antibodies to chemieaUy synthesized peptides. Cell 33:671-682. Tennekoon GL Yoshino J, Peden KWC, Bigbee J, Rutkowsid ~ Kishimoto Y, DeVries GH & McKhann GM (1987) Transfection of neonatal rat Schwarm ceils with SV-40 large T antigen gene under control of the metallothionein promoter. Y Cell Bio1105:2315-2325. Tjian R (1981) T antigen binding and the control of SV40 gene expression. Cell 26:1-2. Van Eldik LI & Zimmer DB (1987) Secretion of S-100 from rat C6 glioma ceils. Brain Res 436:367-370. Voipe Jl, Fujimote K, Mamsa JC & Agrawal HC (1975) Relation of C-6 glial cells in culture to myelin. Biochem 1 152:701-703. White BA & Bancroft FC (1982) Cytoplasmic dot hybridization. J Biol Chem. 257:8569-8572. Wolfe RA, Sato GH & MeClure DB (1980) Continuous culture of rat C6 gtioma in serum-free medium. J Cell Bio187:434-441.
Differential Regulation of Myelin Gene Expression in SV40 T Antigen-transfected Rat Glioma C6 Cells M. Bong, 1 A. Chakrabarti, 1 N. Banik, ~E.L. H o g a n , I M. K a n o h , 2 R . C . W i g g i n s , z and
G. KonaV :~
Received October 18, 1990; accepted January 16, 1991 Rat glioma C6 cells were stably Iransfected with a pSV3-neo plasmid containing SV40 T antigen gene, and geniticin-resistant transfectants (designated C6T ceils) were cloned. The C6T cells grew as well-defined foci of cells showing squamous or irregular morphology. The doubling time for transfected ceils was reduced by approximately 40% as compared to control C6 ceils. The transfecfion with T-antigen also affected the expression of genes coding for structural myelin proteins and for myelin-associated enzymes. The steady-state level of proteolipid protein (PLP)-speeific mRNA in C6T cells was 44% lower than in parental C6 cells. On the other hand, the transfection upregulated the expression of myelin-associated glycoprotein (MAG) by 153%. The activity of 2':3'cyclic AMP phosphodiesterase (CNP) was increased by approximately 80 % in the C6T cells as compared to untransfected, control ceils. The activity of calcium-activated neutral proteinase (CANP) was also significantly elevated in the transfectants by approximately 50% and 220% for millimolar and micromolar form respectively. The results indicate that T antigen affects the expression of myelin genes, although, individual genes appear to be differenflyregulated implying the existence of several independent regulatory mechanisms. KEY WORDS: C6 ceils; transfection; SV40 T antigen; gene expression;
myelin proteins; myelin enzymes.
INTRODUCTION Although, many important advances have been made within the past several years in our understanding of structures and biological properties of major myelin proteins at the 1 Department of Neurology, Medical University of South Carolina, 171 Ashley Ave., Charleston, S.C. 29425 32Departmen~ of Anatomy, West Virginia University School of Medicine, Mqrgantpwn, WV 26506. To whom correspondence should b~ addressed at Department of Anatomy, west virginia University, School of Medicine, 4052 HSN, Morgantown, WV 26505.
7 0~85-7490/91/0300-0007506.50/0 9 1991 Plenum Publishing Corporation
8
Konat et al.
molecular biological level, little is known about the factors that regulate the expression of corresponding genes. Cultured myelinating oligodendroeytes do not proliferate and this severely limits their use for genetic and biochemical studies of the process. The rat glioma C6 cell line is closely related to the oligodendrocyte, and has served as a model for the in vitro investigation of glial properties (Seeds et al., 1970; Clark and Perkins, 1971; Pfeiffer, 1973; Bachrach, 1975; Bennett et al., 1977; McMorris et al., 1985; Van Eldik and Zimmer, 1987; Reggiani et al., 1987; Herschman et al., 1973; Wolfe et al., 1980). Although the C6 cells do not produce myelin-related structures, they express some of the myelin-specific proteins, i.e., 2':3'-cyclic nncleotide 3'-phosphohydrolase (CNP) (McMorris et al., 1985) and proteolipid protein (PLP)(Volpe et al., 1975; Milner et al., 1985). We have found that these cells also express calcium-activated neutral proteinase (CANP)(Banik et al., 1988), an enzyme which is specifically localized in brain myelin (Banik et al., 1987). Recent advances in cell and molecular biological methodology open new possibilities to examine the genetic regulatory mechanisms by incorporation of foreign genes which would perturb the normal expression of the host genes. The aim of the present study was to determine whether the expression of myelin-specific genes in the C6 cells can be experimentally manipulated. The experimental paradigm employed transfection of the C6 cells with Simian Virus 40 (SV40) T antigen gene, which is a potent effector of mammalian gene expression (Tjian 1981; Mitchell et al., 1987).
MATERIALS AND METHODS Cell Culture
The C6 glioma cells were obtained from Dr. Kenneth Leskawa (University of Louisville, KY) and grown in Ham's F-10 medium/Dulbecco modified Eagle's medium (1:1) (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (Biofluids, Inc., Rockville, MD) and 1% of antibiotic-antimycotic mixture (Gibco) in plastic flasks or dishes at 37~ under 96% air/4% C02. For measurement of growth rate, the cells were plated in 6-weU (35 mm) tissue culture plates (Costar, Cambridge, MA) at 16 x 105 cells/well. At different intervals the cells were trypsinized and counted in a hemacytometer. cDNA Probes and Vectors
Plasmid p27 containing full-length eDNA (3.2 kb) for rat PLP (Milner et al., 1985) and plasmid plB236 containing partial eDNA (1.5 kb) for rat MAG (Sutcliffe et al., 1983) were obtained from Dr. Robert Milner (Scripps Clinic, La Jolla, CA). Plasmid pSV3-neo (Okayama & Berg 1983), containing the SV40 T antigen gene and selectable neo gene (providing resistance to geneticin) was obtained from the American Type Culture Collection.
Differential Regulation of Myelin Gene Expression
DNA Transfection
106 cells grown for 24 h in 10 cm plastic dish were transfected with pSV3-neo plasmid by calcium phosphate technique using CellPhect Transfection kit (Pharmacia). The culture was subsequently incubated for 12 hours at 37~ and subjected to osmotic shock by incubation in isotonic HEPES buffer, pH 7.5 containing 15% glycerol for 3 minutes. The ceils were rinsed, incubated in standard culture medium (containing 10% fetal calf serum) for 48 hours, trypsinized, diluted 1:2 and replated on new dishes with fresh medium. 12-14 hours later geneticin sulfate (G418) (Gibco) was added to the medium to a final concentration of 0.5 mg/ml. The drug-containing medium was changed every 4 days. After 14-16 days of selection, individual colonies (geneticin-resistant clones) were picked and transferred to 24-well plates (Costar, Cambridge, MA). The transfected cells, designated C6T, were passaged at confluence, expanded into 75 cm2 flasks and used for further experiments. Mock-transfected (without DNA) cells did not yield any G418-resistant colonies. R N A Isolation
Cells were washed with PBS, scraped and collected in sterile eppendorf tubes, and cytoplasmic RNA was isolated by the procedure of White and Bancroft (1982). The poly (A) + RNA was separated out on oligo(dT)-cellulose column as described by Maniatis et al., (1982). Poly (A)§ RNA was stored in 70% ethanol at -20~ prior to use. The concentration of nucleic acids was estimated by absorbance at 260 nm. Northern and Dot Blot Analysis
PLP and MAG radioactive probes were generated as previously described (Konat et al., 1988). For northern analysis, total RNA (10-30 ~tg) was denatured with formaldehyde, electrophoresed on 1% agarose gel and subsequently transferred in 10X SSC to BIOTRANS nylon membrane (ICN Biochemicals, Cleveland, OH). The membrane was further processed and hybridized with labeled eDNA probes (1-5 x 106 cpm/ml) according to the manufacturer's protocol. The position of the specific messages was detected by autoradiography. For quantitation, the autoradiograms were scanned using a LKB 2202 Ultroscan laser densitometer and the relative amount of radioactivity was determined by measuring the integrated optical density. Dot blot analysis was performed as described by Davis et al.(1986). Serial dilutions were made in 15X SSC, starting with 10 ~g of total RNA or 1-5/I.tg of poly (A)§ RNA denatured with formaldehyde. The samples were applied to BIOTRANS nylon membrane using dot blot apparatus and further processed and quantitated as described for northern blots. Cell Subfractionation and Protein Determination
Cells were washed twice with saline and homogenized in 20 mM Tris-acetate buffer (pH 7.5) containing 1 mM EDTA, 2.5 mM 2-mercaptoethanol, 1 mM PMSF (phenyl methyl sulfonyl fluoride) and lmM NAN3. Following centrifugation of the homogenate at 110,000
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Konatet al.
g for 60 min pellet and supematant (cytosol) fractions were collected. The protein content was determined by the method of Smith et al (1985).
Determination of Enzymatic Activities
Adenosine 2',-cyclic nucleotide 3'-phosphohydrolase (CNP) activity was assayed by the method described earlier (Kurihara and Tsukada, 1967: Banik and Davison, 1969). One unit [U] of the enzyme activity corresponds to one, l&rnoleof 2'-AMP formed per hour. Micromolar calcium-activated neutral protease (gM CANP) and its millimolar isoform (raM CANP) were separated from inhibitor and partially purified by DEAE-cellulose and ~34henyl sepharose chromatography (Murachi, 1984; Chakrabarti and Banik 1988). C-azocasein substrate was prepared by reductive alkylation of azocasein (Sigma Chemical Co., St. Louis, MO) with 14C-formaldehyde (American Radiolabeled Chemicals Inc., St. Louis, MO), and the enzyme activities were assayed as previously described (Banik et al., 1987). Samples containing EGTA (5 mM) served as controls. One unit of activity [lJ] was defined as the amount of enzyme which liberates 103 cpm of acid-soluble radioactivity per hour. RESULTS
The rat glioma C6 cells were stably transfected with a pSV3-neo construct containing the SV40 T antigen gene (Okayama & Berg 1983) and the geniticin-resistant transfectants (designated C6T cells) were cloned. The relatively high transfection frequency of about one transformant in 6 x 104 cells demonstrates that the C6 cells effectively take up recombinant DNA molecules from the calcium phosphate precipitate and stably express its genes. The transfected, C6T cells were more tightly packed together than normal C6 cells and tended to grow over each other forming foci (Fig. 1). Most of the C6T cells showed irregular or squamous morphology and tended to detach from the substratum. These cells grew to a slightly higher saturation densities in monolayer cultures than did parental C6 cells. The doubling time for C6T cells was 11.3 hr as compared to 18 hr for the C6 cells. The transfection also caused doubling in the number of dead cells as compared to the parental cell line. Both C6 and C6T cells expressed myelin-specific protein genes, viz. PLP and MAG as evaluated by steady-state level of specific mRNA messages determined by hybridization technique (Fig. 2). Two PLP transcripts of 3.2 and 1.6 kb were detected by Northern blot analysis, whereas MAG eDNA probe hybridized to single regions of 2.5 kb. The size-pattern of the messages was not affected by the transfecfion of C6 ceils with T antigen, although, pronounced differences in their steady-state level were observed. Thus, the level of PLP messages in the transfectants was only 56% of that of the parental (control) cells (Table I). On the other hand, the transfection elevated MAG-specific mRNAs to 253% of control. The transfection of the C6 cells with T antigen also upregulated the expression of myelin-associated enzymes, i.e. CNP and CANP (Table If). Both total (expressed per 106 cells) and specific (expressed per mg of protein) CNP activity was elevated in C6T cells to
Differential Regulation of Myelin Gene Expression
11
Fig. 1. The morphological changes in rat glioma C6 cells induced by stable transfection with SV40 T antigen. Control (C6) cells (upper panel) and transfeeted (C6T) cells (lower panel) were grown as described in Materials and Methods and phase-contrast photographed at 200X magnification.
12
Konat et aL
PLP
MAG
C
C
T
T
Ftg. 2. ~xpression ot myelin protein genes in C6 ceils and in C6 ceils stably transfected with SV40 T antigen (C6T cells). 25 txg of cytoplasmic RNA were applied per lane and northern blot a n a l y s i s was performed as described in Materials and Methods with eDNA probes for PLP (panel A) and MAG (panel
B).
Table L The Effect of T antigen on PLP and MAG mRNA in the C6 cells. Message PLP MAG The relative messages was northern blot and Methods). + S.D. from 7
% of control C6 Cells 56 + 13 253 + 81 amount of specific determined by dot and analysis (see Materials Values represent means experiments
Differential Regulation of Myelin Gene Expression
13
Table H. The Effect of T antigen on CNP, mM CANP and uM CANP activity in C6 Cells
Enz),me CNP IxMCANP mM CANP
Total acdvit~ (U/106 Cells) C6 cells C6T ceils 301.2 + 12.2 542.2 + 11.4a 79 +- 18 252 • 24 a 474 4- 96 711 + 14IF
Specific Activit~ (U/mg protein) C6 cells C6T cells 54.0 + 6.6 97.2 + 6.2 a 149 + 28 478 • 82a 915 + 142 1373 + 204b
U - units of activity (cf. Materials and Methods). Values from four experiments are expressed as mean + S.D. Significance levels by t-test: a p < 0.001 ; b p < 0.02; c p < 0.05
approximately 180% of the parental cells. The activity (both total and specific) of millimolar and micromolar CANP was increased in the transfectants to 150 and 320% of control value, respectively. Approximately 88% of CNP, 78% of mMCANP and 15% of I.tMCANP activity was associated with particulate fraction of the C6 cell homogenate and the subcellular distribution of these three enzymatic activities was not affected by the transfection (results not shown).
DISCUSSION
The rat glioma C6 cells have been shown to express some of the myelin-specific genes, i.e. CNP (McMorris et al., 1985), PLP (Volpe et al., 1975; Milner et al., 1985) and CANP (Banik et al., 1988). In this communication we show that the C6 cells also express MAG, a myelin-associated glycoprotein thought to play a regulatory role in myelinogenesis (Quarles 1984). The MAG-specific mRNAs from the C6 cells migrate as a single band corresponding to 2.4-2.5 kb. This is the size range of MAG messages found in the rat brain, where the primary transcripts of MAG gene undergo alternative splicing resulting in two messages of 2,389 and 2,474 bp (Lai et al., 1987). Thus, the processing of MAG transcripts in the C6 cells appears (at this resolution) to be the same as in the oligodendrocytes. The size of PLP messages in the C6 cells, observed in this study, are congruent with those reported previously (Milner et al., 1985) and resemble the pattern found in the rat brain. The large T antigen of Simian Virus 40 (SV40) is a multifunctional phosphoprotein, which shares the ability to both activate and repress gene transcription in mammalian cells (Tjian, 1981; Mitchell et al., 1987). Other functions contribute to its ability to promote immortalization as well as neoplastic transformation of mammalian cells (Colby & Shenk 1982; Petit et al., 1983; Chou & Martin 1975; Asselin & Bastin 1985 Lewis & Martin 1979). The molecular mechanisms of these functions are as yet undefined but may be related to the ability of T antigen to form a tight complex with nuclear phosphoprotein pS3 (Crawford et al., 1981), to bind to double-stranded DNAs (Clark et al., 1983), to its protein
14
Konat et al.
phosphorylating activity (Griffin et a/., 1979), and/or to the combination of the above properties. It has been recently demonstrated that myelin-specific genes are also susceptible to the effects of T antigen. Chert et al., (1987) found that the transformation of Schwann cells with SV40 virus upregulates the expression of MAG and CNP genes. On the other hand, Tennekoon et al., (1987) observed downregulation of the expression of CNP by T antigen introduced into the Schwalm cells by transfection. The discrepancy may be due to different experimental paradigms, i.e. viral transformation vs. transfection with recombinant T antigen gene. This study shows that the T antigen also effects myelin genes in the CNS-derived C6 cells. Thus, PLP expression was downregulated to approximately half of its message level in the parental ceils, while the expression of other genes was upregulated in the C6T cells as compared to the control C6 cells (Table I & II, Fig. 3). The message sizes of PLP and MAG were not altered by the transfection with T antigen. Thus, T antigen seems to exert its effect(s) in the C6 cells at the genomic level and not to affect the posttranscriptional processing of the primary transcripts. Although, the mechanisms of regulatory action of T antigen remain unknown, individual genes appear to be differently regulated (reciprocal regulation of expression of PLP versus other genes)(Fig. 3) implying the existence of several independent regulatory mechanisms reflecting different arrays of cis- and trans-acting elements for individual genes. The mechanism of expression of myelin genes, i.e. the relationship between transcription and translation is not fully understood in the C6 cells. The levels of PLP and MAG proteins
350 300 250
:-----..-^" , A A A A f
f3_~ 200 KMMMM
,
15o
o 100 50 0
~KXx~ 9 PLP ~ MAG ~ CNP ~]p~M CANP [ ] mM CANP
Fig. 3. T antigen-induced alteration in the expression of myelin genes in rat glioma C6 cells. The values are calculated from data shown ill Table 1 andTable 2 and are expressed as percent of parental (untransfeeted) cells.
Differential Regulation of Myelin Gene Expression
15
in both parental and transfected C6 cells was below the detection by immunoblot analysis (results not shown). At this point it is not known, whether the mRNAs increase is accompanied by increased synthesis of MAG or, whether PLP synthesis is blocked by T antigen. However, the cropping and dumping of the C6T cells may be explained by an increased expression of MAG on their surface, and thus provide a circumstantial evidence for the translation of the induced messages. Furthermore, the increase in the amount of CNP and CANP (as expressed per either cell or protein, Table II) may indicate that the messages were normally translated in this system. In conclusion, the demonstration that the C6 cells normally express MAG in addition to other major myelin proteins extends the applicability of the C6 cells as a model for oligodendrocytes. Furthermore, the expression of the myelin-specific genes can be experimentally modulated, and hence, these cells provide a useful model system to study the mechanisms of regulation of these genes.
ACKNOWLEDGMENTS The authors wish to thank Dr. Maria Trojanowska for helpful advice and discussion during the course of this work and Ms. Joan Kingsley for secretarial assistance. This work was supported by NIH grants NS-12044, NS-11066, and NS-21353 from the NINDS.
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