Expression of Oligodendrocyte-Associated Genes in Cell Lines Derived from Human Gliomas and Neurohkistoinas1

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(CANCER RESEARCH 53. 170-175. January I. 19931 Expression of Oligodendrocyte-associated Genes in Cell Lines Derived from Human Gliomas and Neurohkistoinas1

Tsuyoshi Kashima, Samuel N. Tiu, Jean E. Merrill, Harry V. Vinters, Glyn Dawson, and Anthony T. Campagnoni2

Menial Relanlalinn Research Center Â¡T.K.. S. N. T.. A. T. C.Â¡,Departments of Neurology Â¡J.E. M.] and Pathology Â¡H.V. V.l. and Brain Research Institute Â¡J.E. M.. H. V. V.. A. T. C.J. University of California Los Angeles. School of Medicine, Los Angeles, California 90024, and Departments of Pediatrics and Biochemical and Molecular Biolog\ 1C. D.Â¡,University of Chicago. School of Medicine. Chicago. Illinois 60637

ABSTRACT readily available, providing a battery of reagents with which to ex amine oligodendrogliomas at the molecular level (5. 6). The use of Two putative human oligodendroglioma cell lines were examined for the expression of the oligodendrocyte-associated genes, T J'-cyclic nucleotide- these markers for in vivo and in vitro studies has resulted in a schema 3'-phosphodiesterase, myelin basic protein, myelin proteolipid proteins, by which oligodendrocytes can be subclassified by differentiation and myelin-associated glycoprotein. The expression of these genes also was state based upon immunocytochemical and molecular biological cri teria (6). Until now, expression of oligodendrocyte (stage)-specific examined in control astrocytoma and neuroblastoma cell lines. In addi tion, the expression of the non-oligodendrocyte-specific genes, glial fibril- genes has not been applied extensively to oligodendrogliomas or cell lary acidic protein (GFAP), neuron-specific enolase and neurofilaments lines derived from them. (NF) NF-L and NF-M also were examined. All the cell lines expressed The purpose of this work was to examine two putative human 2',3'-cyclic nucleotide 3'-phosphodiesterase, neuron-specific enolase, and oligodendroglioma cell lines to determine the extent to which these vimentin, and none expressed myelin-associated glycoprotein. The "oligo- cell lines expressed oligodendrocyte-specific genes. We also com dendrocyte-specific" myelin proteolipid protein mRNAs and the "neuron- pared the expression of these genes in the oligodendroglioma-derived specific" NF-L mRNA were expressed in the two astrocytoma cell lines, cell lines to their expression in cell lines derived from other neural cell which also expressed GFAP. Expression of intermediate filament protein types. In addition to supporting the assignment of these cell lines to genes was more restricted. The astrocytoma, neuroblastoma, and oligo dendroglioma cell lines expressed only GFAP, NF-M, and cytokeratin K7, the oligodendrocyte lineage through their expression of both normal respectively. These results: (a) provide molecular data confirming the and novel markers of oligodendrocyte derivation, we also found that classification of the two cell lines as oligodendrogliomal and suggest that astrocytoma- and, to some extent, neuroblastoma-derived cell lines, expressed "oligodendrocyte-specific" genes. their molecular profiles are indicative of immature oligodendrocytes; i/>i demonstrate the expression of cytokeratins in oligodendrogliomal cell lines and suggest that apparent GFAP expression in oligodendrogliomas de MATERIALS AND METHODS tected by immunocytochemical methods may be due to cross-reactivity with cytokeratins, with which they share common polypeptide sequence; Cell Culture. Tumor cell lines were grown in Iscove's modified Dul- and ici indicate that astrocytoma cell lines can exhibit a "mixed" pheno- becco's medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% type, expressing genes associated with fully differentiated oligodendro fetal calf serum (Sigma, St.Louis, MO), 2 ITIML-glutamine, and antibiotics cytes and neurons. (100 units/ml penicillin, 100 ug/ml streptomycin, and 250 pg/ml Fungizone; Irvine Scientific) at 37Â°Cin 5% CO2. The human brain tumor cell lines used INTRODUCTION were two oligodendroglioma cell lines. HOG (7) and TC620 (8), two astro cytoma cell lines, U251MG and U373MG (9), and one neuroblastoma cell line, The identification of oligodendrogliomas has been based largely SK-N-SH (10). The original TC620 cells were supplied by Dr. L. Manuelidis upon morphological and immunocytochemical criteria ( 1), although (Yale University, New Haven, CT) and subcloned by Dr. J. Merrill (11). relatively few tumors have been studied and immunohistochemical U251MG cells were supplied by Dr. B. Westermark (University of Uppsala, data have not always been available. Interestingly, astrocytomas may Uppsala, Sweden). U373MG and SK-N-SH cell lines were purchased from the show focal regions of oligodendrogliomatous differentiation (2), al American Type Culture Collection (Rockville, MD). though the significance of this is unclear. This association of the two The characteristics of the TC620 cell lines used in this study have been types of differentiation has led to speculation that oligodendrogliomas published previously (8, II). Under our growth conditions the cells had a and astrocytomas might be derived from the transformation of a doubling time of 24 h. somewhat shorter than the astrocytoma lines. They exhibited a bipolar, epithelioid-like morphology with thick, short cytoplasmic common glial precursor or that neoplastic oligodendrocytes might transform into astrocytomas giving rise to mixed gliomas (3, 4). processes; and a high nucleusxytoplasm ratio with the nuclei generally con The oligodendrocyte is the myelin-forming cell in the central ner taining a single nucleolus. The cells have been reported to express surface galactocerebroside, thereby placing them in the oligodendrocytic lineage (11). vous system and it expresses the genes coding for the myelin struc Their expression of surface components, determined by immunocytochemical tural proteins in a cell-specific and temporally regulated fashion (5). analysis, is characteristic of immature oligodendrocytes (II). However, characterization of oligodendrogliomas with markers of Some characteristics of the HOG oligodendroglioma cell line have been mature oligodendrocyte gene expression has met with mixed success, published recently (7). These cells grew well under our growth conditions, since tumors may be derived from immature or precursor oligoden exhibiting a doubling time of 18 h. Morphologically, these cells were also drocytes or by the time of examination the tumor cells may have lost epithelioid-like with a bipolar and in some cases triangular form. Occasionally, the phenotype of the mature cell. the cells exhibited long, thin processes, particularly when grown at low density. Biochemical markers (protein, nucleic acid, and lipid) of immature The nucleusxytoplasmic ratio in these cells was also very high. These cells have been reported to express the oligodendrocytic markers galactocerebro and precursor stages of oligodendrocyte differentiation have become side, galactocerebroside sulfate, and CNPase.' Northern Blot Analysis. Total RNA was isolated by the method of Chom- Received 7/2/92: accepled 10/19/92. czynski and Sacchi (12) from the cortex of a 4-5-month-old human and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with confluent human tumor cells. Total RNA was selected for polyadenylated RNA 18 U.S.C. Section 1734 solely to indicate this fact. 1This research was supported by NIH Grants NS23022 (A. T. C), NS23322 (A. T. C>, 'The abbreviations used are: CNPase. 2'.3'-cyclic nucleotide _V-phosphodiesterase: NS263I2 (H. V. V.), and HD25831 (A. T. C, J. E. M.) and National Multiple Sclerosis Society Grants PPOI27 (J. E. M.). RG2233-A-I (A. T. C), and RG2319-A-6 (J. E. M.). cDNA. complementary DNA; SDS. sodium dodecyl sulfate: SSPE, saline-sodium phos- 2 To whom requests for reprints should be addressed, at Mental Retardation Research phate-EDTA: GFAP. glial fibrillary acidic protein; MAG, myelin-associaled glycoprotein; Center. 47^*48 NPI. UCLA School of Medicine. 760 Westwood Plaza. Los Angeles. CA MBP. myelin basic protein: NF. neurofilament protein: NSE. neuron specific enolase; PLP. 90024. myelin proteolipid protein: IF, intermediate filament. 170

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1993 American Association for Cancer Research. CHARACTERIZATION OF CNS TUMOR CELL LINES by chromatography on oligodeoxythymidylate cellulose (Collaborative Bio- cDNA hybridized to the expected 2.2-kilobase mRNA, the product of medical Products. Bedford, MA) (13). Polyadenylated RNA (7 ug/lane) was the major MBP gene promoter, in RNA isolated from human cortex electrophoresed in denaturing agarose gels containing 2.2 Mformaldehyde and (Fig. 1, Lane I), but it did not hybridize to a 2.2-kilobase RNA in the transferred to nylon membrane filters (Nytran: Schleicher & Schuell. Inc.. cell lines. Instead, the probe hybridized to two MBP gene-related Keene. NH) with lOx standard saline-citrate (Ix concentration is 150 DIM 2.6- and 5.5-kilobase mRNAs in some of the cell lines. The HOG cell sodium chloride-15 msi sodium citrate) (14). The RNA was immobili/ed onto filters by UV-cross-linking at 160 J/nr after drying them at 80Â°C.cDNA line (Fig. 1, Lane 3) expressed both the 2.6-kilobase MBP mRNA and probes were labeled to a specific activity of 1-3 X |0g dpm/ug with [Ã•2P-]- the 5.5-kilobase MBP-related mRNA. The TC-620 cell line (Fig. 1, Lane 4) expressed only the 5.5-kilobase MBP-related mRNA. In dCTP (3000 Ci/mol: DuPont, NEN. Boston. MA) by random priming (15). The blots were hybridized at 42Â°Cin 50% formamide, 5% SDS. 25 ITIMsodium one of the astrocytoma cell lines (U251MG; Fig. 1, Lane 5) the phosphate (pH 7.2). I ITIMEDTA. and 10 ug/ml denatured low molecular 2.6-kilobase mRNA appeared to be expressed, while in the other weight DNA for at least 15 h and washed once in 2X SSPE/0.1% SDS at room (U373MG; Fig. 1, Lane 6). both MBP gene-related mRNAs were temperature for 15 min, twice in IX SSPE/0.1% SDS at 37Â°Cfor 15 min, and expressed very faintly. There was no detectable expression of these twice in 0.1 x SSPE/0.1% SDS at 55Â°Cfor 15 min. The blots were exposed to RNAs in the neuroblastoma cell line (Fig. 1, Lane 7). The expression X-ray film. XAR-5 (Kodak. Rochester, NY) with an intensifying screen at of these two mRNAs occurs earlier in the developing mouse brain -80Â°C (14). in vivo (30)4 and in culture (31)4 than the 2.2-kilobase MBP mRNAs Western Blot Analysis. Cell pellets and frozen tissues were homogenized transcribed from the major promoter of the MBP gene. in 10% SDS buffer (50 mg tissue/ml) and protein concentrations were deter Like the 2.2-kilobase MBP mRNA. PLP is expressed later in de mined by the modified method of Lowry et al. (16) (Sigma: protein assay). Fifty ug of protein of each sample were electrophoresed in SDS-poly- velopment, in the mature oligodendrocyte. Although PLP mRNA was acrylamide gels (17). Proteins were transferred to nitrocellulose membranes not detected in the Northern blots of the two oligodendroglioma (Schleicher & Schuell) by the method of Towbin et al. (18). Specific antibodies (Fig. 1, Lanes 3 and 4) and the neuroblastoma (Fig. 1, Lane 7) cell and membranes were incubated in phosphate-buffered saline containing lines, surprisingly, PLP/DM20 mRNA of the appropriate size (3.2 0.5% Blocking reagent (Boehringer Mannheim. Indianapolis, IN) at room kilobases) was expressed at low levels in the astrocytoma cell lines, temperature for at least 10 h. Bound antibodies were detected with 15-20 U251MG and U373MG (Fig. 1, Lanes 5 and 6 ). We could not detect uCi/blot 125I-protein A ( 10-15 mCi/mg protein) (DuPont, NEN), washed three the expression of MAG mRNAs in any of the tumor cell lines (data times with phosphate-buffered saline containing 0.1% Triton X-100, and ex posed to X-ray film at -80Â°C. not shown). Expression of Neuronal-specific mRNAs. Two 2.8- and 1.8- Probes and Antibodies. cDNA probes were obtained from the following sources: rat CNPase (19) and rat MAG cDNAs (20) were obtained from Dr. kilobase bands were evident in the Northern blots of all the cell lines David Colman: human MBP (21) and PLP (22) cDNAs were isolated in our probed with the NSE cDNA (Fig. 2). The 2.8-kilobase band is the size own laboratory; rat NSE cDNA (23) was obtained from Dr. Gregor Sutcliffe: of authentic NSE mRNA and the 1.8-kilobase band is likely to rep a full-length mouse GFAP cDNA was isolated by us from a mouse brain library resent non-neuronal enolase mRNA based upon its size and cross- with a clone provided to us by Dr. Nicholas Cowan (24): the human GFAP hybridization characteristics (23). Although all the cell lines expressed cDNA was obtained from Dr. Steven Reeves (25); a human vimentin cDNA both enolase mRNAs, the non-neuronally derived cell lines expressed was purchased from the American Type Culture Collection (26): and the a higher proportion of the non-neuronal enolase mRNA. In the neu human NF-L and NF-M cDNA clones were the gifts of Dr. Virginia M-Y. Lee roblastoma cell line the proportion of NSE of non-neuronal enolase (27). Of the ten cDNA probes used, four were of rodent origin. In all cases the mRNA was much higher than in the other lines. probes used recognized mRNAs of the appropriate size in control human tissue We also examined the expression of the NF-L and NF-M mRNAs lanes run in all Northern blots and shared significant homology with the human in the cell lines using human NF-L and NF-M cDNA probes (Fig. 2). homologues. Anti-CNPase polyclonal antibodies were the gift of Dr. David Colman; The 3.5-kilobase NF-M mRNA was most abundant in the neuroblas anti-GFAP polyclonal antibodies were purchased from Biomedicai Technol toma (Fig. 2, Lane 7) and was expressed to a lesser extent in the ogy, Inc.; anti-vimemin monoclonal antibody (clone 3B4) and anti-cytokeratin human cortex (Fig. 2, Lane ÃŒ).Itwas not expressed in the cell lines K7 monoclonal antibody (clone CK 7) were purchased from Boehringer- of glioma origin. Surprisingly, the 2.2- and 4.0-kilobase NF-L tran Mannheim: and anti-NSE polyclonal antisera were purchased from Poly- scripts were detected not only in the neuroblastoma (Fig. 2, Lane 7) sciences, Inc. and human cortex (Fig. 2, Lane 1) but also in the astrocytoma cell lines (Fig. 2, Lanes 5 and 6). There was no expression of either RESULTS neurofilament mRNA in the oligodendroglioma-derived cell lines (Fig. 2, Lanes 2 and 3). In these studies several markers of cells in the oligodendrocyte Expression of the Astrocyte-specific GFAP mRNA and the Re lineage were examined. Markers of mature oligodendrocyte gene ex lated Intermediate Filament Gene, Vimentin. Upon short exposure pression included MBP, PLP, and the MAG. Markers of earlier stages (3 h) of the Northern blots to a mouse cDNA probe, a 3.5-kilobase of oligodendrocyte gene expression were CNPase and the 2.6- and human GFAP mRNA was evident only in the human cortex and the ~5-kilobase MBP gene-related mRNAs (28, 29). Markers of other two astrocytoma cell lines (Fig. 3, Lanes I. 5, and 6, respectively). neural cell types included GFAP for astrocytes as well as NSE and After longer exposures ( 18 h), however, two other bands of 2.0 and 1.7 NF-L and NF-M for neurons. Vimentin expression also was examined kilobases were evident in the HOG and TC620 oligodendroglioma cell because of its relationship to GFAP, a member of the same interme lines (Fig. 3, Lanes 3 and 4). The 2.0-kilobase band also could be seen diate filament protein subclass. In addition to the HOG and TC620 in the neuroblastoma cell line upon longer exposure (Fig. 3, Lane 7). oligodendendroglioma cell lines we examined the expression of these Although the 1.7 kilobase and 2.0 kilobase RNAs were expressed markers in the astrocytoma-derived cell lines, U251MG and in the HOG and TC620 oligodendroglioma cell lines, we could U373MG, and the neuroblastoma-derived cell line, SK-N-SH. not determine if it also was expressed in the astrocytoma cell lines because of the spreading of the GFAP band with increased expo Northern Blot Analysis of the Human Oligodendroglioma- sure time. Furthermore, because vimentin, an intermediate filament derived Cell Lines, HOG and TC620 protein, belongs to the same subclass as GFAP and shares a 66% Expression of Oligodendrocyte-specific mRNAs. The rat CN homology at the nucleotide level with GFAP mRNA in their coding Pase cDNA probe hybridized to the 2.6-kilobase CNPase mRNA in all the human neurogenic tumor cell lines (Fig. 1). The human MBP 4 Manuscript in preparation. 171 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1993 American Association for Cancer Research. CHARACTERIZATION OF CNS TUMOR CELL LINES

CNPase MBP PLP 1 234567 1 34567 1234567

- 5.5kb

-2.6kb 3'2W>- 9 -2.6kb 2.2kb-

Fig. 1. Northern blot analysis of polyadenylated RNA (7 (jg/lane) isolated from tumor cell lines and human cortex probed with oligodendrocyte-associated cDNAs. The same blot was hybridized, sequenlially. lo cDNA probes for CNPase. MBP. and PLP. Lane I. human cortex: Lane 2. blank; Lane 3. HOG; Lane 4, TC620; Lane 5. U251MG; Lane 6. U373MG; Lane 7, SK-N-SH. Left: A single 2.6-kilobase band corresponding to CNPase mRNA was detected in all the cell lines and human cortex. Middle: The 2.2-kilobase (kh) MBP mRNA was evident in human cortex after a short exposure time of 6 h (Lane /). The 2.6- and 5.5-kilobase mRNAs of the MBP-related gene were most prominent in the HOG line (Lane 3) and to lesser extents in the other glioma cell lines (Lanes 4-6) after an extended exposure of 72 h. Right: A 3.2-kilobase band corresponding to the PLP/DM20 mRNA was detected in the human cortex (Lane /) and the two astrocytoma cell lines (Lanes 5 and 6).

NSE NF-L NF-M 1 234567 1234567 1234567 -4.0kb -3.5kb -2.8kb -2.2kb -1.8kb

Fig. 2. Northern blot analysis of mRNA from the cell lines with neuronal-specific genes. The same blot shown in Fig. I was stripped and reprobed with cDNA probes for NSE (left}, NF-L (middle), and NF-M (right). Lane I. human cortex; Lane 2. blank; Lane 3. HOG; Lane 4. TC620; Lane 5, U251MG; Lane 6. U373MG; Lane 7. SK-N-SH. Left: The 2.8-kilobase (kb) NSE mRNA was detected in all the cell lines. In addition, a 1.8-kilobase non-neuronal enolase mRNA was clearly evident in the glioma cell lines (luanes 3-6). Middle: Heavy bands at 2.2 and 4.0 kilobases. corresponding to the sizes of the NF-L mRNAs. were detected in the neuroblastoma (Lane 7) and the two astrocytoma (Lanes 5 and 6) cell lines, and to a lesser extent in the human cortex. There was no evidence of these mRNAs in the two oligodendroglioma cell lines (Lanes 3 and 4). Right: A single 3.5-kilobase NF-M mRNA was detected in human cortex (Lane 1) and in the neuroblastoma cell line (Lane 7). A lower molecular weight, cross-hybridizing band were expressed in the two astrocytoma cell lines (Lanes 5 and ft) regions (25, 26), we wondered if the 2.0-kilobase band could be posed in order to detect the CNPase bands in the cell lines. The Mr vimentin mRNA. 46,000 CNPase isoform was expressed in the TC620 (Fig. 4, Lane 3), We prepared cDNA libraries of the two oligodendroglioma cell U251MG (very faintly; Fig. 4, Lane 4), U373MG (Fig. 4, Lane 5) and lines, screened them with the mouse GFAP cDNA, and isolated sev the neuroblastoma (Fig. 4, Lane 6) cell lines. The A/r 48,000 isoform eral distinct cDNA clones. The clones were sequenced and the se of CNPase was expressed in the neuroblastoma (Fig. 4, Lane 6) and, quences were compared with others filed in the GENBANK and possibly, the TC620 (Fig. 4, Lane 3) cell lines. EMBL databases. Such an analysis revealed that one of the clones We could detect no MBP polypeptides in the cell lines although the corresponded to a full-length human vimentin mRNA and hybridized antisera readily revealed MBP isoforms in the human cortex and specifically to the 2.0-kilobase band in the Northern blot shown in Fig. mouse brain samples (data not shown). We were also unable to detect 3. The vimentin mRNA appeared to be expressed in all the tumor cell the presence of the PLP/DM20 polypeptides in any of the cell lines lines examined. with several antisera, even those in which PLP mRNAs were clearly We also isolated a cDNA clone (GRG-6) which, upon analysis, evident. It is possible that the lack of immunoreactivity was due to indicated that it encoded human cytokeratin K7 (32). We then sub- differences between the posttranslational fatty-acylation patterns in cloned a portion of the GRG-6 clone that was specific to cytokeratin the cell lines and in vivo, possibly altering the ability of the antibody K7 mRNA and probed Northern blots with it (Fig. 3). The resulsts to recognize the antigen. The detection of PLP/DM20 mRNAs by indicated that cytokeratin K7 mRNA expression was restricted to the Northern blots and the lack of detection of the polypeptide products in oligodendroglioma-derived cell lines (Fig. 3, Lanes 3 and 4), and it cell lines and primary cultures have been observed by others (30). was not expressed in the astrocytoma or neuroblastoma-derived cell However, it is also possible that the mRNAs for these myelin proteins lines (Fig. 3, Lanes 5-7). are transcribed in the cell lines but not translated. Western Blot Analysis of the Human Glioma-derived Cell Lines, Expression of the authentic M, 51,000 human GFAP polypeptide and some lower molecular weight immunoreactive products appeared HOG and TC620 to be restricted to the two astrocytoma cell lines (Fig. 5, top, Lanes 4 Expression of Oligodendrocyte-specific RNAs. The presence of and 5) and human cortex (Fig. 5, Lane I ) when the Western blots were two CNPase polypeptide isoforms with molecular weights of 46,000 exposed for 18 h. On the other hand, when the blots were exposed for and 48,000 was detected in samples of human cortex by Western blot 72 h, higher molecular weight. anti-GFAP immunoreactive bands analysis, with the Mr 46,000 isoform predominating, although this is were clearly evident in the oligodendroglioma cell lines and to a not evident in Fig. 4 (bottom. Lane I) because the film was overex slightly lesser extent in the neuroblastoma cell line. We did not detect 172

GFAP Keratin K7 Vimentin short exposure long exposure 1234567 34567 34567 34567

-3.5kb

2.0kb- -2.0kb 1.7kb- -1.7kb

Fig. 3. Northern blot analysis of mRNA from the cell lines with IF-related gene cDNAs (GFAP. keratin K7. and vimentinl. Both a long ( 18-h) and short (3-h) exposure of the GFAP blot is shown. Lane I. human cortex; Lane 2. blank; Lane 3. HOG: Urne 4. TC620; Lane 5, U251MG; Lane 6, U373MG; Lane 7. SK-N-SH. GFAP panels: The 3.5-kilobase (kb) GFAP mRNA was detected in human cortex (lane /) and the two astrocytoma cell lines (Â¡Mnes5and f>)at short exposure times. At longer exposures, additional 2.0-and 1.7-kilobase mRNAs became evident in the other cell lines (C.K., Ã•Mnes3, 4. and 7). Keratin ptint'l: Cytokeratin K7 mRNA was detected exclusively in the oligodendroglioma cell lines (Lanes 3 and 4} when the blots were hybridized to a K7-specific probe. Vimenrin panel: The 2.0-kilobase vimentin mRNA was expressed in all the cell lines.

expression. We thought that oligodendroglioma-derived cell lines might be useful models of cells in the oligodendrocyte lineage "ar rested" at specific developmental stages, much as cells derived from NSE lymphomas and leukemias have served as useful models to further our 46K- understanding of normal B- and T-cell ontogeny (31, 33). Unfortu nately, few oligodendroglioma cell lines have been analyzed with respect to their expression of oligodendrocyte stage-specific genes; therefore we undertook such an analysis of two such cell lines. In order to examine the specificity of gene expression in the HOG -. Ã€ and TC620 oligodendroglioma lines we compared their patterns of gene expression with two established astrocytoma- and one estab lished neuroblastoma-derived cell lines. The pattern of gene expres Fig. 4. Western blot analysis of the cell lines for expression of neuron-specific enolase and CNPase proteins. Fifty ug of total protein per lane were electrophoresed in SDS- sion in the two oligodendroglioma cell lines generally reflected what polyacrylamide gels and electrotransferred to nitrocellulose membranes. The proteins might be expected of cells in the oligodendrocyte lineage. However, were detected by specific poly- and monoclonal antibodies, and the antigen-antibody complexes were visualized by I25l-labeled protein A and autoradiography ( I week), l^ine our analyses of the cell lines, in general, revealed some interesting and I. human cortex; Lane 2. HOG; Lane 3. TC620; Lane 4. U251MG; Lane S. U373MG; unanticipated results. Lane f>.SK-N-SH. Top: The M, 46.IXX) NSE polypeptide was expressed by all the cell lines. Boitnm: The two CNPase M, 46.IXX)and M, 48.000 Â¡soformsare merged into one 1 large band in human cortex because the gel was overexposed to reveal hands in the cell lines. The M, 46.000 CNPase isoform was expressed in the TC620 (Lane 3), U251MG (very faintly. Lane 4), U373MG (htnc 5) and the neuroblastoma (Initie ft) cell lines. The - 51K Mr 48.000 isoform of CNPase was expressed in the neuroblastoma (Ã•Miie6) and, possibly, the TC620 (lane 3i cell lines. K. thousands. GFAP * the presence of authentic M, 51,000 GFAP, even after extended ex posure, in the oligodendroglioma or neuroblastoma-derived cell lines (data not shown). Expression of the related intermediate filament - 58K protein, vimentin (MT58,000), was observed in all the tumor cell lines, Vimentin along with the presence of lower molecular weight immunoreactive products in the astrocytoma cell lines (Fig. 5. bottom. Lanes 2-6). Additional evidence for the expression of cytokeratin K7 in the HOG and TC620 cells was provided through Western blot analysis of Fig. 5. Western blot analysis of the cell lines for the several intermediate filament proteins. Each lane contained 50 ug of total protein. Proteins were detected by anli-GFAP the cell lines with a cytokeratin K7-specific monoclonal antibody polyclona! antibody and anti-vimentin monix'lonal antibody (panel B) with exposure limes (Fig. 6, Lanes 2 and .?). The Mr 55.000 cytokeratin K7 protein was of Â¡8hr. Â¡MneI. human cortex; Lane 2. HOG; Lane 3. TC620; Lane 4. U251MG; Lane 5. U373MG; Lane f>.SK-N-SH. Top: The M, 51.(XX) GFAP and several lower molecular clearly expressed in the two oligodendroglioma cell lines, consistent weight cross-reactive polypeptides were evident in the two astrocytoma cell lines (/^mr.v with the expression of the cytokeratin K7 mRNA. but its expression 4 and 5) and in the human cortex (hinc /). fiottoni: The Mr 58.(XX) vimentin polypeptide could not be detected in the other cell lines examined. was detected in all the cell lines along with the presence of lower molecular weight Similarly, we detected the neuron-specific enolase MT 46,000 cross-reactive products in ihe astrocyloma cell lines. polypeptide in human cortex and in all of the tumor cell lines (Fig. 4, top. Lanes 1-6). consistent with the presence of the NSE mRNA observed on the Northern blots (Fig. 2). Tables 1 and 2 summarize the Northern and Western blot data in order to permit easy comparison of the gene expression patterns of the Keratin tumor cell lines. K7 â€”¿55K

DISCUSSION

We undertook this study, initially, to determine the extent to which Fig. 6 Western blot analysis of the cell lines for cytokeratin K7. Each lane contained 50 ug of total protein. Proteins were detected by anli-cytokeratin K7 monoclonal antibody transformed cell lines isolated from oligodendrogliomas might serve with an exposure time of 18 h. Â¡Mne1.human cortex; Ijme 2, HOG; iMne 3. TC620; Ijiue as models with which to study stage-specific oligodendrocyte gene 4. U25IMG; Â¡Mne5. U.173MG; Lane f>.SK-N-SH. 173 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1993 American Association for Cancer Research. CHARACTERIZATION OF CNS TUMOR CELL LINES

Table I mRNA expression (Northern hhtl in human central nen'ous system tumor cells (oligodendrocytes) in vitro (40) and in immature oligodendro cell lines cytes of human fetal spinal cord in vivo (41). In addition, there have CVII lines been several reports of GFAP immunoreactivity in pure oligodendro- Size mRNA (kilobases) HOG TC620 U251MG U373MG SK-N-SH gliomas (4, 42, 43). In this study we found that two cell lines derived CNPase 2.6 from human oligodendrogliomas express cytokeratin K7, which MBP 2.2 shares polypeptide homology with GFAP. This conservation is suffi MBP-related 2.6 mRNA 5.5 ciently strong at both the polypeptide and nucleotide levels that we PLP/DM20 3.2 could use a mouse GFAP cDNA probe to isolate a human cytokeratin MAG 2.5 cDNA. We also found that anti-GFAP antibodies reacted with other, Vimentin 2.0 NSE 2.8 higher molecular weight proteins on Western blots, although not as NF-L 2.2 readily as with authentic GFAP. The IF protein family has a conserved 4.0 a-helix coiled-coil region in the rod domain (44). In fact, an anti-IF NF-M 3.5 GFAP 3.5 common antigen monoclonal antibody has been produced that recog K7 keralin 1.7 nizes the end of the 2B segment in the rod domain common to all members of the IF protein family in many species (45, 46). These Table 2 Protein expression (Western blot) in human centrai nervous system tumor findings raise a question about the molecular identity of the GFAP cell lines immunoreactivity observed in immature oligodendrocytes and oligo linesS dendrogliomas. In view of the potential for cross-immunoreactivity i7Â«.(M,) ProteinCNPaseMBPPLP/DM20GFAPVimentinK7U373MG46,000HOG TC620 U251MG among members of the IF family, it is possible that the antigens being recognized might be vimentin, cytokeratin. or some other member of +48.000 + Â±17.000-21.500027,00020.00051,000Â± the IF protein family. It underscores the need to use additional mo lecular criteria beyond the immunocytochemical identification of an tigens in such situations. +58.000 + +55.000 + + + keratinNSECell +46.000 + ACKNOWLEDGMENTS + + + +SK-N-SH_+---+-+ The authors are especially grateful to Dr. Paul Zeltzer for many stimulating discussions and for his comments on the manuscript. We are also grateful to Dr. The patterns of gene expression, summarized in Tables I and 2, Joyce Benjamins for suggesting the line of research that led to the identifica suggested that certain neural genes were expressed in all the trans tion of cytokeratin K7 in the oligodendroglioma cell lines. formed cell lines regardless of origin. These included CNPase, vimentin, and NSE. Neuron-specific enolase is considered to be a neuronal marker (34) and CNPase is generally considered to be oli- REFERENCES godendrocyte specific, yet in this study all the cell lines expressed 1. Rubinstein, L. J. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1993 American Association for Cancer Research. Expression of Oligodendrocyte-associated Genes in Cell Lines Derived from Human Gliomas and Neuroblastomas

Tsuyoshi Kashima, Samuel N. Tiu, Jean E. Merrill, et al.

Cancer Res 1993;53:170-175.

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