Human Glial Fibrillary Acidic Protein

[CANCER RESEARCH 51. 1553-1560. March 1. 1991] Human Glial Fibrillary Acidic Protein: Complementary DNA Cloning, Chromosome Localization, and Messenger RNA Expression in Human Glioma Cell Lines of Various Phenotypes1

Erik Bongcam-Rudloff, Monica Nister, Christer Betsholtz, Jia-Lun Wang, Goran Stenman, Kay Huebner, Carlo M. Croce, and Bengt Westermark2 Department of Pathology, I'nirersity Hospital, S-751 85 Uppsala, Sweden fE. B-R., M. .V, C B., J-L. H'.,B. H'.];Department ofOral Pathology, I'nirersity of GÃ¶teborg, Sweden f(j. S./: and Fels Institute for Cancer Research and Molecular Biology, Temple I'nirersity School oj Medicine. Philadelphia, Pennsylvania 19140 [K. H., C. M. C.]

ABSTRACT notypes, we have in the present investigation cloned a full length human GFAP cDNA and determined the corresponding amino Glial fibrillar) acidic protein (GFAP) is a constituent of intermediate filaments of glial cells of the astrocyte lineage. \Vecloncd a human GFAP acid sequence. The chromosomal location of the GFAP gene complementary DINA,deduced the amino acid sequence, and established was also established. the chromosomal location (17q21) of the GFAP gene by Southern blot Recent studies have suggested that PDGF has a physiological hybridization of somatic cell hybrids and by in situ hybridization. The role in the development of glial cells in the central nervous authenticity of the complementary DNA was proven by expressing it in system. Thus, the bipotential O-2A progenitor cells of the rat glioma cells lacking endogenous GFAP; after microinjection of the com optic nerve respond by proliferation to PDGF produced by type plementary DNA, such cells became positive for staining with GFAP 1 astrocytes (11, 12). Given the fact that the \-sis oncogene antibodies. The levels of fibronectin (FN) and GFAP mRNA of ten encodes a PDGF-like growth factor (13-15), the finding that human glioblastoma cell lines, determined by Northern blot hybridization intracerebral injection of simian sarcoma virus in newborn of RNA. were related to other phenotypic characteristics (cell morphology and expression of the genes encoding platelet-derived growth factor marmosets gives rise to brain tumors of the glioblastoma phe (PDGF) receptors). A high expression of GFAP mRNA was found only notype (16) suggests that an autocrine activation of the PDGF in cells lacking fibronectin mRNA and protein. Glioma cells with a receptor in glial cells is a causal event in tumorigenesis. fibroblastic phenotype (bipolar, FN*/GFAP~) were found to express both Whether a similar autocrine mechanism also operates in the types of PDGF receptors (a and 0). Relatively high levels of PDGF a- genesis of human "spontaneous" glioblastoma is unclear. There receptor mRNA, in the absence of /3-receptor expression, were found in are several examples of human glioma cell lines that express cell lines that express GFAP and lack detectable levels of fibronectin one or both of the PDGF chains (17-19) concomitantly with mRNA. The findings are compatible with the idea that the genes encoding receptors for PDGF (18-20), but it is not known if activation PDGF receptors in glioma cells are regulated in concert with other genes, of these genes is a cause or consequence of malignant transfor the expression of which may reflect the developmental program of normal mation. PDGF synthesis and expression of the cognate receptor glia cell lineages. may occur in certain normal glia cells (cf., Refs. 12 and 21), and this phenotype may be retained after malignant transfor INTRODUCTION mation. We have reasoned that if this were the case, one should Intermediate filaments are 8-10-nm fibers that form part of expect that expression of PDGF and its receptors be related to the cytoskeleton of virtually all cell types in higher eukaryotes other phenotypic markers. This is a difficult problem to study (1-4). On the basis of their tissue-restricted distribution, inter since, particularly in the human, very few differentiation mark mediate filament proteins have been divided into five main ers for glial cells are available, and the progenitor cell for classes: desmin; vimcntin; GFAP,1 neurofilament proteins; and glioblastoma has not been identified. keratins. All intermediate filament proteins have the same In the present investigation we have used the best character structural organization with a central Â»-helical rod domain ized astrocyte marker, namely GFAP, and studied its expression in relation to cell morphology and expression of PDGF Â«-and flanked by amino and carboxy termini of variable sizes. /^-receptor and fibronectin. GFAP is a constituent of glial cells of the astrocytic lineage (5) and is generally also present in primary glial brain tumors (6). However, a high proportion of cell lines established from MATERIALS AND METHODS malignant gliomas do not express GFAP (7). Immunohisto- chemical analyses of glioma cell lines have shown that the cDNA Cloning and Nucleotide Sequence Analysis. Standard molecu expression of GFAP and the extracellular matrix protein fibro lar biology techniques (22) were used unless otherwise indicated. The mouse GFAP cDNA clone Gl (23) was radiolabeled using ["PJdeox- nectin (8) is mutually exclusive (9), even in cells derived from ynucleotidc triphosphate and the Klenow fragment of DNA polymerase the same tumor (10). In order to facilitate further studies on I. The radiolabeled plasmid was used to screen a cDNA library made the expression of GFAP in human glial cells of different phe- from the human clonal glioma cell line U-342 MGa C12:6 (24) which Receivcd 8/27/90; accepted 12/17/90. expresses GFAP (10). The recombinant phages were plated on Esche- The costs of publication of this article were defrayed in part by the payment richia coli C600 hfl (25). Duplicate nitrocellulose filter lifts were of page charges. This article must therefore be hereby marked advertisement in hybridized with 32P-labeled Gl at 42Â°Cin 20% formamide-5 x SSC (1 accordance with 18 I'.S.C. Section 1734 solely to indicate this fact. 1This work was supported by grants from the Swedish Cancer Society and the x SSC is ISO m,M sodium chloride-15 HIM sodium citrate)-50 mivi N1H (CA 25875 and C'A .19860). sodium phosphate. pH 7.0-5 x Denhardt's solution-0.1% SDS-200^g/ 2To whom requests for reprints should be addressed. ml sonicated single stranded DNA and washed in 0.5 x SSC-0.1 % SDS ' The abbreviations used are: GFAP. glial fibrillar}- acidic protein: cDNA. at the same temperature. Positively hybridizing clones were plaque complementary DNA: PDGF. platelet-derived growth factor: SSC. standard saline-citrate; SDS. sodium dodecyl sulfate; MEM, Eagle's minimum essential purified and XDNA was isolated. The cDNA insert was isolated by medium: PBS. phosphate-buffered saline: poly(A)" RNA. polyadenylated RNA: EcoRI digestion and subcloned in pUC-13. The nucleotide sequence of kbp. kilobase pairs: FN. fibronectin. the human cDNA restriction fragments was determined by the dideox- 1553

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1991 American Association for Cancer Research. HUMAN GLIAL FIBRILLARE Ai II) PROTEIN ynucleotide chain termination method (26) after subcloning into M13 after hybridization were at 40Â°C.Slides were exposed for 8-16 days at phage derivatives (27). 4Â°Cand subsequently developed and G-banded using 0.25% Wright's Analysis of the Expression of Microinjected GFAP cDNA. To deter stain in 0.6 M phosphate buffer (pH 6.8) for 5-15 min. The chromo mine the expression of cloned GFAP cDNA. the clone E2C2 (see somal positions of grains located on chromosomes or touching chro below) was inserted in sense orientation into the EcoR\ site of the mosomes were plotted onto ideograms of G-banded human chromo retroviral vector RD2 (kindly provided by Dr. L. Hellman. Department somes at the 550-band stage (35). of Immunology, University of Uppsala). The expression plasmid was Cell Lines and Culture Conditions. The establishment of cell lines designated phGFAP-RD2. The human U-343 MG glioma cell line, from human malignant gliomas has been described (36). The cell lines which does not express GFAP ( 10), was grow n to subconfluence on 18- used in the present investigation originated from tumors that were x 18-mm glass coverslips in Falcon dishes in MEM supplemented with diagnosed as glioblastoma multiforme. All cell lines were grown as 10% fetal calf serum (Gibco Ltd., United Kingdom) and antibiotics. monolayer cultures in Eagle's MEM supplemented with 10% newborn phGFAP-RD2 DNA, at a concentration of 0.5 ^g/ml, was microin- calf serum (GIBCO) and antibiotics (100 units of penicillin and 50 Â¿ig jected into the cell nucleus using an Olympus IMT2-SYF injectoscope. of streptomycin per ml). Cultures were maintained at 37Â°Cin humidi The cells were then incubated at the same culture conditions for 48 h. fied air containing 5% CO;. By using phase contrast microscopy we Cells were fixed in 3% paraformaldehyde in PBS for 10 min at room assigned the cell lines to either of five morphology groups (19): epithe- temperature and in 100% acetone for 1 min at -20Â°C.Immunostaining lioid; polygonal; pleomorphic/astrocytoid: fascicular; or bipolar/ of GFAP was performed using a rabbit immunoglobulin to cow glial fibroblastic. fibrillar) acidic protein (Dakopatts, Denmark) following a rhodamine- Northern Blot Analysis of Human Glioma Cell Lines. Cells were conjugated swine anti-rabbit IgG antibody (Dakopatts) at room tem grown to confluency in routine medium on 100 mm plastic Petri dishes perature. Cells were rinsed with phosphate-buffered saline and exam or 850-cnr Falcon roller bottles. Total cellular RNA was isolated using ined by fluorescence microscopy. Cells not treated with injections of the LiCI/urea method. mRNA was enriched by poly(A)* selection on GFAP cDNA growing on the same coverslip were used as a control. oligodeoxythymidylate cellulose (Pharmacia). Ten /jg of poly(A)* RNA Chromosomal Localization of the Human GFAP Gene. The GFAP were denatured for 5 min at 65Â°Cin 50% formamide. 2.2 M formalde cDNA clone pCIA-5 described in this report (see below) was used to hyde, 20 niM morpholinepropanesulfonic acid (pH 7.0), 5 mivisodium determine the chromosomal localization of the human GFAP gene. acetate, l mM EDTA, and 10% glycerol; run on 0.8% agarose-formal- Cloned DNA probes for genes which have previously been regionally dehyde gels: transferred to nitrocellulose filters: and baked at 80Â°Cfor localized on chromosome 17 were used for comparison and are de 4 h. cDNA probes were ':P-labeled by random priming. Hybridization scribed in detail in Table I. Probes (entire plasmid including insert and (lO^cpm/ml) was performed in 50% formamidc-5 x SSC-5 m\i EDTA- vector) were radiolabeled by nick-translation using [l:P]deox>nucleotide 0.5% SDS, 1 x Denhardt's. 100 f/g/ml salmon sperm DNA at 42Â°C triphosphates to a specific activity of 1 x IO9 cpm/Mg: 1 x 10* cpm for 12 h and washing in 0.5% SDS-0.1 x SSC at 50Â°Cfor 2 h. Kodak were used for each Southern blot hybridization. XAR-5 film was used for exposure at -70Â°C. Isolation, propagation, and characterization of parental cells and The following human cDNA probes were used for Northern blot somatic cell hybrids have been described (28). For regional localization hybridization analyses: (a) the GFAP cDNA probe pCIA-5 as described of the GFAP gene on chromosome 17, a series of hybrid cell lines in this communication: h) the PDGF /^-receptor cDNA probe (Table 1). each retaining only a partial chromosome 17. were used (29. pHPDGFR-2A3 (37): (c) the PDGF Â«-receptor cDNA probe 30). pHPDGFR Al (38); (il) the fibronectin probe pHFNl consisting of a Cellular DNA was isolated as described (31). digested with an excess 380-base pair Pst\ insert in pBR322 and encoding the active cell of restriction endonuclease Hind\\\ or Sstl, size fractionated in 0.8% attachment domain of fibronectin (39). agarose gels, transferred to nylon membrane using 0.4 M NaOH (32), Indirect Immunofluorescencc Staining. Glioma cells were suspended in Eagle's MEM-10% fetal calf serum, seeded sparsely on coverslips and dried under vacuum. Prehybridization and hybridization were carried out in 1.5 x saline-sodium phosphate-EDTA (1 x saline-sodium contained in 35-mm Petri dishes, and allowed to grow for 2-3 days. phosphate-EDTA is 0.15 M NaCI-0.01 M NaH2PO4-l mM disodium Before the first incubation, as well as between each subsequent incu EDTA, pH 7.4), i% SDS, 0.5% nonfat powdered milk, and 0.5 mg/ml bation, cells were washed 3 times in PBS. Incubations were made at carrier salmon sperm DNA at 42Â°C.Filters were washed with 0.1 x room temperature in a moist chamber. Rabbit anti-fibronectin (1:50) SSC-0.1% SDS at 68Â°Cand exposed to Kodak XAR-5 films at -80Â°C (kindly provided by Dr. Antti Vaheri) was allowed to react for 30 min. with intensifying screens for 12-16 h. and bound antibodies were visualized with rhodamine-conjugated goat For in situ chromosomal hybridization. pCIA-5 was nick-translated anti-rabbit immunoglobulin (1:50. 30 min) (Dakopatts). Cells were with [3H]dTTP to a specific activity of 2.4 x IO7 cpm//ig. Normal fixed with 3% paraformaldehyde in PBS for 20 min at room tempera human melaphase chromosomes from one male and two females were ture and with 100% acetone for 1 min at -20Â°C and then incubated used for hybridization. The methods for in situ hybridization and with the monoclonal GFAP-3 antibody (1:100) (kindly provided by Dr. autoradiogruphy were as described (33, 34). Hybridization was carried Peter Collins) for 30 min. The antibody was visualized by a fluorescein out at a concentration of 100 ng labeled probe/ml hybridization solu isothiocyanate-conjugated anti mouse immunoglobulin (1:20. 30 min) tion, and with a 1000-fold excess of carrier herring sperm DNA. Washes (Dakopatts).

Table 1 Regional localisation oj the GFAP %eneon chromosome 1? The SKBR3 cell line carries an amplified HKR-2/nrÂ« locus (59) indicated by ++++: the amplification unit involves several other 17-linked genes" bul not the GFAP gene (not shown). The region of chromosome 17 carried by hybrids cl 31 and 275S has been defined cytogenelicallv (60) and confirmed using DNA probes for chromosome 17-linked genes (28. 31. 60-62) as shown here and elsewhere (28. 30. 63). The region of chromosome 17 retained in hybrids N9 and cl9 has been defined using the DNA markers listed in here and by others (60. 63). The GFAP cognate sequences segregate with the Hox-2 gene in these hybrids: thus the GFAP cognate locus is distal to the NiiFR and proximal to the PKCâ€”locus. probesHer-2/Neu 17-linkcd DNA

PKC-n<17ql2-K|2l) NGFR Hox-2 GFAP 17 region CelllineSKBR

' K. Unebner, unpublished results. 1554

l MetGluArgAroAra IleIhrSerAlaAlaArgArgSerTyrValSer 16 l ATGGAGAGGAGACGCATCACCTCCGCTGC!CGCCGCTCCTACGTCTCC 48 MERRRITSAARRSYVSS GLATORRLGPGT]RTSLARMPPPLP(TJRVDFSLAGALNÂ¿h 17 SerGlyGluMetMetValGlyGlyLeuAlaProGlyArgArgLeuGly 32 MERRRVlTSAARRSYVSal Â¡RRLGPGPRLSLARMPPPLPARVOFSLAGALNT p 49 TCAGGGGAGATGATGGTGGGGGGCCTGGCTCCTGGCCGCCGTCTGGGT 96 33 ProGlyThrArgLeuSerLeuAlaArgMetProProProLeuProThr 48 97 CCTGGCACCCGCCTCTCCCTGGCTCGAATGCCCCCTCCACTCCCGACC 144 GFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAEL head *â€”Â¡â€”Â»rod 49 ArgvalAspPheSerLeuAlaGlyAlaLeuAsnAlaGlyPheLysGlu 64 GFKETRASERAEMMELNDRFASYIEK 145 CGGGTGGATTTCTCCCTclcCTGGGGCACTCAATGCTGGCTTCAAGGAG 192 GFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAEL 65 ThrArgAla SerGluArgAlaGluMetMetGluLeuAsnAspArgPhe 80 193 ACCCGGGCCAGTGAGCGGGCAGAGATGATGGAGCTCAATGACCGCTT 240 Ã•RELIRELRLRLDQLTANSARLEVERON rLRQKLOOElKlRLEA 81 Ala SerTyr IleGluLysValArgPheLeuGluGinGinAsnLysAla 96 241 GCCAGCTACATCGAGAAGGTTCGCTTCCTGGAACAGCAAAACAAGGCG 288 RELRLRLDO.LTANSARLEVERO^RELt rLROKLQDEff_RLEADNNIAAYRQÃ•ADEATEftNLAAYRQEADEAT 97 LeuAlaAlaGluLeuAsnGinLeuArgAlaLysGluProThrLysLeu 112 289 CTGGCTGCTGAGCTGAACCAGCTGCGGGCCAAGGAGCCCACCAAGCTG 336 QOEIRTlh 113 AlaAsp ValTyrGinAlaGluLeuArgGluLeuArgLeuArgLeuAsp 128 337 GCAGACGTCTACCAGGCTGAGCTGCGAGAGCTGCGGCTGCGGCTCGAT 384 LÃ‡IRjJm

129 GinLeuThrAlaAsn SerAlaArgLeuGluValGluArgAspAsnLeu 144 QYE/ H|SKFADLTOAA 385 CAACTCACCGCCAACAGCGCCCGGCTGGAGGTTGAGAGGGACAATTTG 432 145 AlaHisAspLeuAlaThrLeuArgGinLysLeuGinAspGluThrLys 160 433 GCACACGACCTGGCCACTCTGAGGCAGAAGCTCCAGGATGAAACCAAG 480 QYE/ RJSKFADLTDAA

177 AspGluAlaThrLeuAlaArgLeuAspLeuGluArgLys IleGluSer 192 GTNESLERQHREQEERHAREAASYOEAl[t)iLEEEGQSLKDEMARHL(JEYCgLLNVKLALD 529 GATGAAGCCACCCTGGCCCGTCTGGATCTGGAGAGGAAGATTGAGTCG 576 GTHESLERQHREQEERHARElUASYQEALARlEEEGQSLKlElEHARHLQEYQOLLNVKLALD 193 LeuGluGluGlu IleArgPheLeuArgLys IleHisGluGluGluVal 208 577 CTGGAGGAGGAGATCCGGTTCTTGAGGAAGATCCACGAGGAGGAGGTT 624 lEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEV 209 ArgGluLeuGinGluGinLeuAlaArgGinGinValHisValGluLeu 224 625 CGGGAACTCCAGGAGCAGCTGGCCCGACAGCAGGTCCATGTGGAGCTT 672 IEIATYRKLLEGEENRIT1PVQTFSNLQIRETSLOTKSVSEGHLKRN--VKTVEMRDGEV

225 AspValAlaLysProAspLeuThrAlaAlaLeuLysGlu IleArgThr 240 lEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEV 673 GACGTGGCCAAGCCAGACCTCACCGCAGCCCTGAAAGAGATCCGCACG 720 241 GinTyrGluAlaMetAlaSerSerAsnMetHisGluAlaGluGluTrp 256 IKESKQEHKD-VM-J h 721 CAGTATGAGGCAATGGCGTCCAGCAACATGCATGAAGCCGAAGAA TGG 768 IKESKQEHKD-\JE p 257 TvrHis SerLysPheAlaAspLeuThrAspAlaAlaAlaArgAsnAla 272 769 TACCAC TCCAAGTTTGCAGACCTGACÃ•GACGCTGCTGCCCGCAACGCA 816 273 Glu LeuLeuArgGinAlaLysHisGluAlaAsnAspTyrArgArgGin 288 Fig. 2. Comparison of the amino acid sequences (single-letter code) of the 817 GAGCTGCTCCGCCAGGCCAAGCACGAAGCCAACGACTACCGACGCCAG 864 human (A), murine (m). and porcine (p) GFAP. The human sequence was deduced 289 LeuGinSerLeuThrCysAspLeuGluSerLeuArgGlyThrAsnGlu 304 TTGCAGTCCTTGACCTGCGACCTGGAGTCTCTGCGCGGCACGAACGAG 912 from the GFAP cDNA clone E2C2 described in this work. The murine sequence 865 was deduced from genomic DNA as described (41 ). The amino acid sequences of 305 SerLeuGluArgGinMetArgGluGinGluGluArgHisValArgGlu 320 two stretches, including the NH; terminus, of porcine GFAP were obtained from 913 TCCCTGGAGAGGCAGATGCGCGAACAGGAGGAGCGGCACGTGCGGGAG 960 Ref. 40. 321 AlaAla SerTyrGinGluAlaLeuAlaArgLeuGluGluAspGlyGin 336 961 GCGGCCAGT TATCAGGAGGCACTGGCTCGGCTGGAGGAAGATGGCCAA 1008 337 SerLeuLysAspGluMetAlaArgHisLeuGinGluTyrGinAspLeu 352 truncated in the 5' end, we rescreened the cDNA library with a 1009 AGCCTCAAGGACGAGATGGCCCGCCACTTGCAGGAGTACCAGGACCTG 1056 5' 120-base pair Sstl-Accl fragment from pCIA-5. The clone 353 LeuAsnValLysLeuAlaLeuAsp IleGlu IleAlaThrTyrArgLys 368 1057 CTCAATGTCAAGCTGGCCCTGGACATCGAGATCGCCACCTACAGGAAG 1104 E2C2 was isolated and found to have a 1.4-kilobase insert, the rod Â«â€”.â€”Â»tail 369 LeuLeuGluGlyGluGluAsnArg IleThr IleProValGinThrPhe 384 nucleotide sequence of which is shown in Fig. 1. The insert is 1105 CTGCTAGAGGGCGAGGAGlAACCGGATCACCATTCCCGTGCAGACCTTC 1152 1409 base pairs long. The putative translation-initiating codon 385 SerAsnLeuGin IleArgGluThrSerLeuAspThrLysSerValSer 400 at the 5' end specifies a 1296-base pair open reading frame 1153 TCCAACCTGCAGATTCGAGAAACCAGCCTGGACACCAAGTCTGTGTCA 1200 which is flanked by a 110-base pair 3'-untranslated sequence. 401 GluGlyHisLeuLysArgAsn IleValValLysThrValGluMetArg 416 1201 GAAGGCCACCTCAAGAGGAACATCGTGGTGAAGACCGTGGAGATGCGG 1248 The NH:-terminal sequence of porcine GFAP has been deter 417 AspGlyGluVal IleLysGluSerLysGinGluHisLysAspValMet 432 mined previously by amino acid sequence analysis of purified 1249 GATGGAGAGGTCATTAAGGAGTCCAAGCAGGAGCACAAGGATGTGATG 1296 protein (40). As can be seen in Fig. 2, the deduced sequence of 433 â€” 1297 TGAGGCAGGACCCACCTGGTGGCCTCTGCCCCGTCTCATGAGGGGCCC 1344 the human GFAP amino terminus is virtually identical to that of porcine GFAP. Thus, the assumed open reading frame of 1345 GAGCAGAAGCAGGATAGTTCGTCCGCCTCTGCTGGCACATTTCCCCAG 1392 the human GFAP cDNA shown in Fig. 1 is likely to be authentic. An alignment with the deduced amino acid sequence 1393 ACCTGAGCTCCCCACCG of mouse GFAP (41) shows an almost perfect match from Fig. 1. Nuclcotidc sequence of the human GFAP cDNA clone E2C2 and the deduced amino acid sequence. The position of the (Â»-helicalrod domain was residue 29 of the human sequence (Fig. 2). The NH;>-terminal determined according to procedures described in Ref. 64. of this residue, the mouse sequence (41), diverges entirely from the porcine and human GFAP sequences. Inspection of the nucleotide sequence of the presumed 5'-untranslated segment After being washed with PBS, coverslips were mounted in glyeerol- paraphenylcnediamine (Sigma) and were viewed in a Lett/, epifluores- of the mouse gene (40), however, shows the presence of an out eence microscope with oil immersion objectives. The relative intensity of frame ATG which corresponds to the initiating methionine of staining on each cell line was estimated (- to +++++). in the human sequence and an 80% identity of the following 45 downstream nucleotides. RESULTS All intermediate filament proteins share the same basic struc ture with a central Â«-helicalrod domain of some 310 amino Isolation of Human GFAP cDNA. A human glioma cell line acids that are flanked by NH2-terminal and COOH-terminal (U-343 MGa Cl 2:6) cDNA library was screened with the mouse head and tail segments which differ in length (1-4). The loca GFAP cDNA probe Gl. Several positive clones were found. tion of these domain structures in GFAP is shown in Fig. 1. One clone, CIA-5, containing a 1.1-kilobase insert was chosen The authenticity of the human GFAP cDNA was further for further characterization. Since this cDNA apparently was examined by subeloning into the expression vector RD2 and 1555

7.2 â€”

Fig. 3. Immunofluorescencc staining of recombinant GFAP expressed in microinjected lJ-343 MG cells. A fibrillary network is decorated by the GFAP antibodies only in areas containing cells microinjected with the GFAP expression vector. Fig. 5. Southern blot analysis of ///m/Ill-digested DNA from mouse (Lane I). human T-cell line (Lane 2), hybrid cl9 (Lane 3). hybrid GL5 (Lane 4). hybrid introduction by microinjection into U-343 MG cells, which N9 (Lane 5). hybrid 275s (LaÃ±e6). hybrid c4 (Lane 7), hybrid G5 (Lane 8), and hybrid BD3 (Lane V) hybridized to the human GFAP probe. The 20-kbp fragment lack endogenous GFAP expression (see below). Indirect im- present in DNA from Lanes 2 to 6 is diagnostic for (he human GFAP gene. munofluorescence staining with GFAP antibodies revealed a Hybrid DNAs in Lanes 3-6 contain whole or parts of human chromosome 17. while hybrids in Lanes 7-9 do not retain any chromosome 17 material (cf. Fig. 4 typical network of GFAP fibers in a fraction of the microin and Table I). jected cells (Fig. 3); cells in adjacent areas served as controls and were devoid of immunostainable fibers. Chromosomal I.ocali/ation of the Human GFAP Gene. A GFAP specific fragment of 20 kbp and numerous (at least 10 panel of cellular DNAs derived from 20 well-characterized are clear on longer exposure) more weakly hybridizing bands rodent-human cell hybrids retaining overlapping subsets of of various lower molecular weights; these bands most likely human chromosome regions (see Fig. 4) was tested for retention represent cross-hybridizing members of a gene family, while of the GFAP gene by Southern blot analysis. An example of the strongly hybridizing band at 20 kbp is the cognate gene. such a Southern blot, after Hind\\\ digestion of control and Lanes 3-6 (Fig. 5) contain DNA from hybrids which contain hybrid DNAs, is shown in Fig. 5. Lane 1 contains mouse DNA the GFAP cognate gene at 20 kbp. while hybrids in Lanes 7-9 and shows only one obvious band (approximately 7.2 kbp). do not retain the cognate gene. The cross-hybridizing bands Hind\\\ cleaved human DNA (Fig. 5, Lane 2) shows one major seen in human DNA do not appear in any of the hybrid DNAs in Lanes 3-9 (or in hybrid cl31, which retains an entire chro

Human Chromosomes <-> mosome 17; not shown) and thus do not segregate with the cognate gene. Since the hybrid in Lane 3 (Fig. 5) retains human HybridBO3 Y chromosome 17 as its sole human chromosome and other G5 positive hybrids (Fig. 5, Lanes 4-6) also retain chromosome 17 Be or a region of 17 while hybrids in Lanes 7-9 do not retain 3a cl31 chromosome 17, the GFAP cognate gene must be linked to GL5 chromosome 17 (cf. Fig. 4). Similar analysis of the entire S3 chromosome panel confirmed linkage of GFAP to chromosome 77-31 DSK20 17 (cf. Fig. 4). Hybrid and control DNAs were similarly ana PBSa lyzed after cleavage with restriction enzyme Sst\ with similar C4 results. G FA P-specific Ssfl fragments of 2.8, 1.2, and<0.5 kbp, 77-28 AB3 representing the cognate GFAP gene, segregated concordantly CI21 (and with a specific region of human chromosome 17) in the 2S5 hybrid panel, while at least six cross-hybridizing Sstl fragments N9 were not linked to chromosome 17. EF3 c/2 Analysis of the somatic cell hybrids retaining specific regions 275S of chromosome 17 (see legend to Fig. 4) was also the basis for 77-3012345678910n|i2[i3141516|17|18I19â€¢â€¢1â€¢Jâ€¢â€¢202122X the regional localization of the cognate GFAP gene on chro Fig. 4. Presence of the human GFAP locus in a panel of 20 rodent-human cell mosome 17. as summarized in Table 1. Since the cognate locus hybrids. â€¢hybrid named in the left column contains the chromosome indicated segregates with the Hox-2 locus in these hybrids (see Table 1), in the upper row: B. presence of the long arm (or part of the long arm. indicated by a smaller fraction of stippling) of the chromosome shown above the column: the GFAP locus is distal to the NGFR locus and proximal to B. presence of the short arm (or partial short arm) of the chromosome listed the PKCâ€”locus and located at 17q21. above the column. D. absence of the chromosome shown above the column. The The regional localization of the human GFAP locus was also column for chromosome 17 is boldly outlined and stippled to highlight correlation of the presence of this chromosome (or region of this chromosome) with the determined by in situ hybridization. A total of 105 metaphase presence of the GFAP gene. The pattern of retention of the GFAP gene in the cells were scored of which 35 (33'V ) had grains on chromosome panel is shown in the column to the right of the figure where the presence of the gene in the hybrid is indicated by a stippled box Â»itha plus sign and absence of 17. Specific labeling was observed over the midregion of the the gene is indicated by an open bo.\ enclosing a minus sign. long arm with a distinct peak at band 17q21. Forty-six% of 1556

total grains on chromosome 17 clustered at this band. A rep ABCDEFGHIJK resentation of the grain distribution on chromosome 17 is shown in Fig. 6. In conclusion, the in situ hybridization data assign the GFAP locus to band 17q21 and thus confirm the data obtained by Southern blot analysis of cell hybrids. Expression of Fibronectin and GFAP in Human Glioma Cell Lines. The levels of fibronectin and GFAP mRNA were studied by Northern blot analysis of poly(A)+ RNA isolated from ten GFAP established human malignant glioma cell lines (Fig. 7; Table 2). This panel of cell lines includes three sublines derived from the U-343 glioma (U-343 MG. U-343 MGa Cl 2:6, U-343 MGa 31L) (18, 42). The fibronectin probe hybridized to a 6-kilobase transcript whereas the GFAP cDNA revealed a 2.7-kilobase mRNA. Fibronectin mRNA was found in four of the cell lines (U-251 MG sp, U-1242 MG, U-343 MG, U-178 MG). GFAP mRNA was found in five of the ten lines analyzed (U-343 MGa 31L, U-343 MGa Cl 2:6, U-251 MG sp, U-373 MG, U-1231 â€žPDGFR A MG). (The 2.7-kilobase band seen on the blot in U-343 MGa 31L hybridized with fibronectin cDNA represents a signal remaining from the previous hybridization with GFAP cDNA). The pattern of mRNA expression of fibronectin and GFAP in the different cell lines was found to conform with the expression of protein, as estimated by indirect immunofluorescence stain ing (Table 2). PDGF a- and ÃŸ-ReceptormRNA in Human Glioma Cells: Relation to Cell Morphology and Levels of Fibronectin and GFAP mRNA. Northern blots of poly(A)+ RNA from the ten PDGFRB glioma cell lines were hybridized with PDGF a- and /i-receptor cDNA probes (Fig. 7). The strongest signal of the 6.5-kilobase PDGF Â«-receptor transcript was found in U-343 MGa 31L; detectable levels were also found in U-373 MG, U-178 MG, U- 343 MG, and U-1242 MG after longer exposure times. Abun dant levels of the 5.4-kilobase PDGF /i-receptor mRNA were found in U-178 MG, U-343 MG, U-1242 MG, and U-1231 MG; a weak signal was found in U-1240 MG. The weak signal seen with the PDGF ^-receptor probe on Northern blot of U- 343 MGa 31L cells represents cross-hybridizing to Â«-receptor FN mRNA (cf. RÃ©f.38). The data on mRNA expression of fibronectin, GFAP, and PDGF a- and /i-receptor were compiled in Table 2 and related to cell morphology as described (19). A striking feature is the essentially inverse relation between the expression of GFAP and fibronectin genes, as has previously been found with regard to the corresponding proteins (9, 10). One exception was found (U-251 MG sp), in which a fibronectin mRNA signal of medium Fig. 7. Northern blot analysis of mRNA from human glioma cells. Ten Mgof strength was found together with GFAP mRNA and protein poly(A)* RNA per lane were electrophoresed in agarose gels and blotted on to nitrocellulose filters which were sequentially hybridized to human cDNA probes expression. Table 2 also makes it clear that glioma cell lines for GFAP. fibronectin. and PDGF <>-(PDGFR A) and Â¿-receptors(PDGFR B). expressing both types of PDGF receptor mRNA. have a fibro- Lane.-I. AG 1523: Lane B. U-343 MGa 31L: Lane C. U-251 MG sp: Lane D, U- blastic phenotype. i.e., bipolar, fibroblast-like morphology and 1240 MG: Lane E. U-1242 MG; Lane F. U-343 MG: Lane G. U-178 MG: Lane H. U-563 MG; Lane I. U-373 MG: Lane J. U-1231 MG; Lane K, U-343 MGa Cl 2:6.

high levels of fibronectin mRNA. Relatively high levels of GFAP Â«-receptor mRNA, in the absence of/i-receptor mRNA expression, were found in two cell lines (U-343 MGa 3IL, U- 373 MG); these belong to the group of cell lines that express GFAP and lack detectable levels of fibronectin mRNA.

DISCUSSION We have cloned human GFAP cDNA and deduced the pri mary structure of the protein. The translation product appears Fig. 6. Representation of the grain distribution on chromosome 17 assigning to be a 432-residue protein with 88 and 93% sequence similar the GFAP locus to region I7q2l by in situ hybridization. ity, respectively, with the two stretches of porcine GFAP that 1557

Table 2 Phenol) pic characteristics of human glioma cell lines A panel of human glioma cell lines of different morphologies were used. Kor original references and assignment to morphological groups see Refs. IX. 19. and 42. The relative amounts of GFAP, fibronectin, and PDGF <>-and iJ-receptor mRNAs were estimated (- to +++++) from sequentially hybridized Northern blots. When the level of expression was extremely low and the cell line difficult to group as positive or negative it is noted as -+. Relative amounts of GFAP and fibronectin (- to +++++) Â»ereestimated from immunofluorescence stainings. Several of the GFAP-positivc cultures contained a minor component of GFAP-negative cells (+/-). PDGFmRNA FN

CelllineU-1231 mRNA+++++ Immunofluorescence mRNA Immunofluorescence mRNA+____+_++++++++++ MGU-343 +++/-++++ 12:6U-343MGa C â€”+++++ ++++ LU-373MGa 31 ++++ +++ n.d. MGU-251 *'/iii 11 i MGspU-I240MGU-563 ++/ -t-4- 4-/( â€”H)â€”(-+) MGU-178 -+ MGU-343 +++++++ +++ MGU-I242MGMorphologyFpilhelioidPolygonalPleo/Astro"Pleo/AstroFascicularFascicularPolygonalBip/FibrBip/FibrBip/FibrGFAP++++++ ++++ +++++ +PDGFjl-receptor Â°Pleo/Astro, pleomorphic/astrocytoid: Bip/Fibr. bipolar, fibroblastic: n.d.. not determined. were previously subjected to amino acid sequence analysis (40). tors. Various kinds of fibroblasts in culture, however, express The authenticity of the cDNA was confirmed by its expression both types of receptors. in microinjected cells. An immunostainable, fibrillary network The present investigation showed a wide range of Â«-and tf- was apparent in transfected U-343 MG cells that lack endoge receptor mRNA levels in human glioma cells, as determined by nous GFAP expression. Northern blot hybridi/.ation analysis. A comparison of PDGF During the progress of the present work. Reeves et al. (43) receptor expression with cell morphology and fibronectin/ reported on the cloning of human GFAP cDNA: our present GFAP phenotype revealed an interesting pattern. One subgroup data confirm their coding sequence. was formed by cells with a fibroblastic phenotype: such cells An alignment with the assumed coding sequence of the mouse displayed the same pattern of PDGF receptor expression as gene showed that the predicted initiating ATG (Met) in the bona fide fibroblasts; i.e., they expressed both types of receptor mouse sequence corresponds to ACG at positions 41-43 in the mRNA. One might argue that these cells are actually derived human nucleotide sequence (Fig. 1). Inspection of the assumed from a separate mesenchymal component, present in the orig 5'-untranslated sequence of the mouse gene (41) shows the inal tumor. The high frequency of establishment of glioma cell presence of a short open reading frame, in which the first 13 lines with such phenotype argues against this notion. Another codons are almost identical to the 5' end of the human coding argument is the finding of the same unique marker chromosome sequence. Since the NHi terminus of mouse GFAP has not in the "fibroblastic" cell line U-343 MG, having the GFAP~/ been established by amino acid sequence analysis, we do not FN* phenotype, and the "glial" line U-343 MGa and clones know at present if the assumed initiating ATG of the mouse thereof, which display the GFAP+/FN phenotype. These cell gene is the authentic one. If this is indeed the case, the conser lines were derived from different primary cultures of the same vation of a sequence, that is untranslated in the mouse and glioma biopsy. Since all derivatives have an inversion in chro most probably translated in the human, is remarkable. mosome 1 [inv(l)pl3q43] (42. 54) they most probably have the Southern blot analysis of human/mouse hybrid cell lines and same clonal origin. Unfortunately, this finding does not abso in situ hybridization were used to assign the human GFAP gene lutely prove a monoclonal origin, since we lack data on the to chromosome 17, band q21. Interestingly, there are several karyotype of the host tissue. It is also interesting that other other genes of interest in relation to GFAP that are located on studies have indicated that certain normal glial cells produce the proximal half of chromosome 17. These include the erbBI fibronectin (55, 56). The possible relationship between these (rat neuro/glioblastoma-derived oncogene homologue) gene, cells and the fibronectin-positive glial cells remains to be the NGFR (nerve growth factor receptor) gene, and the NF1 elucidated. (von Recklinghausen neurofibromatosis) gene (44). Several A relationship between the pattern of PDGF receptor expres members of the cytokeratin gene family have also been mapped sion and glioma cell phenotype was further evidenced by the to this region of chromosome 17 (44). Recent mapping studies, finding that cell lines expressing GFAP had no detectable, or assigning the mouse GFAP gene to chromosome 11 (45), very low, levels of PDGF /3-receptor mRNA. In this group we indicate that GFAP is part of a large syntenic gene cluster found two cell lines (U-343 MGa 31L and U-373 MG) that including at least 22 genes located on human chromosome 17 expressed relatively high levels of PDGF Â«-receptor mRNA. and mouse chromosome 11 (44, 45). Expression of Â«-receptors in U-343 MGa 31L has been de Recent studies have shown that there exist two types of scribed previously; these cells were used for initial characteriza PDGF receptors that differ in ligand specificity. The PDGF Â«- tion (20) and cDNA cloning (38) of the Â«-receptor. receptor binds all three isoforms of PDGF (PDGF-AA, -AB, Our study highlights the phenotypic heterogeneity of glioma -BB) with high affinity, whereas the /i-receptor binds only cell populations, with regard to cells both within a certain PDGF-BB with high affinity (46, 47). The two receptors are population and between different populations. Cellular hetero structurally homologous (20. 37,48), and both have been show n geneity is not a specific trait of gliomas and is considered an to transduce mitogenic signals in response to the proper ligand important character of malignant tumor cell populations in (49, 50). Available information suggests that the Â«-and fl- general inasmuch as it may form the basis for tumor progression receptors are differentially expressed in normal cells. Thus, the (see Ref. 57 for review). According to Nowell's original hypoth O-2A glial progenitor cell of the rat opticus nerve expresses esis (58), phenotypic heterogeneity may be the result of the Â«-receptors only (51), whereas human meningeal cells (52) and genetic instability of tumor cells allowing for the generation of rat brain capillary endothelial cells (53) express only /i-recep- a plethora of genotypic and phenotypic variants. Another, not 1558

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1991 American Association for Cancer Research. Human Glial Fibrillary Acidic Protein: Complementary DNA Cloning, Chromosome Localization, and Messenger RNA Expression in Human Glioma Cell Lines of Various Phenotypes

Erik Bongcam-Rudloff, Monica Nistér, Christer Betsholtz, et al.

Cancer Res 1991;51:1553-1560.

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