[CANCER RESEARCH 60, 1221–1224, March 1, 2000] Advances in Brief Fibroblast Growth Factor Receptor-1 -Exon Exclusion and Polypyrimidine Tract- binding Protein in Glioblastoma Multiforme Tumors 1 Wei Jin, Ian E. McCutcheon, Gregory N. Fuller, Eileen S-C. Huang, and Gilbert J. Cote 2 Departments of Medical Specialties [W. J., E. S-C. H., G. J. C.], Neurosurgery [I. E. M.], and Pathology [G. N. F.], The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 Abstract Neoplastic transformation of glial cells alters inclusion of the  exon in human fibroblast growth factor receptor-1 (FGFR-1) mRNA transcripts. Although normal cells predominantly include the  exon, this exon is excluded in most glioblastoma cell transcripts, creating a high-affinity receptor form. In this study, we identified polypyrimidine tract-binding protein (PTB) as a regulator of FGFR-1 splicing. PTB interacted in a sequence-specific manner with the ISS-1 regulatory element in the intron upstream of the  exon. PTB expression was also strongly increased in seven malignant glioblastoma multiforme tumors relative to adjacent normal tissue, but not in a low-grade astrocytoma. These results suggest that increased expression of PTB may contribute to glial cell malignancy. Introduction Alternative recognition of the  exon during processing of FGFR-1 3 RNA produces receptor forms that vary in their affinity for fibroblast growth factor (1, 2). Because FGFR-1 plays a primary role in many cell growth and differentiation pathways, precise regulation of its RNA splicing is critical. However, normal recognition of the  exon is altered during the malignant progression of glial cells, pro- ducing a receptor form lacking the  exon and with enhanced affinity for fibroblast growth factor (3, 4). Expression of this form of FGFR-1 in glial cells is believed to provide a cell-growth advantage and possibly to contribute to glial cell malignancy (3). Using a cell culture model, we previously identified two intronic RNA sequences flanking the  exon, ISS-1 and ISS-2, that are required for glioblastoma cell-specific FGFR-1 RNA splicing (5, 6). Deletion or mutation of either of these elements reverses the splicing phenotype observed in glioblastoma cells so that the FGFR-1 mRNA includes the  exon. In this study, we found that the trans-acting factor PTB specifically bound to the upstream element ISS-1 and was overexpressed in patient glioblastomas, suggesting that PTB may regulate glioblasto- ma-specific FGFR-1 RNA splicing. Materials and Methods Cell Culture and Tissue Specimens. The human astrocytoma cell line SNB-19 and the human choriocarcinoma cell line JEG-3 were maintained as described previously (7). Tissue samples were obtained from 10 patients who underwent therapeutic removal of primary or recurrent brain tumors at M. D. Anderson Cancer Center. Histopathological examination showed the samples to be glioblastoma multiforme (n = 7), anaplastic astrocytoma (n = 1), or low-grade astrocytoma (n = 1). One patient with a prior history of oligoden- droglioma had radionecrosis when a lesion mimicking a tumor was resected. Tissue samples were obtained from white matter adjacent to each tumor and deemed normal based on gross histological appearance. Plasmid Constructs. The plasmid constructs pFGFR-17, pFGFR-D1, pF- GFR-M2, and pFGFR-M4 have been described previously (5). The plasmid constructs used for UV cross-linking were obtained by the TA cloning of inserts into the vector pGEMT Easy according to manufacturer’s protocol (Promega Corp., Madison, WI). The inserts were created by PCR amplification of pFGFR-17, pFGFR-M2, and pFGFR-M4, using primers FP109 (5'- GGAAATGAGGGCCCATCCGCTT-3') and FP110 (5'-CCTCCAAAAAGT- CAAAGG-3'). The final constructs, pFGFR-67, pFGFR-68, and pFGFR-69, respectively, were obtained by an ApaI digestion and religation to remove the multilinker sequences. The ligation sites of plasmid constructs were sequenced to confirm the identity of each clone. RNA Isolation and RT-PCR. The transfection of cell lines, RNA isola- tion, and RT-PCR analysis were performed as described previously (7). Total RNA was isolated from 100 mg of normal and tumor tissue by sonication in Catrimox-14 (Qiagen, Chatworth, CA) as described previously (8). Because of the presence of nonspecific amplification bands, the RT-PCR protocol used to amplify tissue-derived RNA was modified from a previously described pro- cedure to include two amplification steps (9). Briefly, reverse transcription was performed with the FGFR-1-specific primer Endo-R using 5 g of total RNA in a 20-l reaction volume. A first round of 11 cycles of PCR (1 min at 94°C, 1 min at 55°C, and 2 min at 72°C) was performed with 10 l of the cDNA and the primers Endo-F and Endo-R (9) in a final volume of 50 l. This was followed by a second round of 17 cycles of PCR (1 min at 94°C, 1 min at 66°C, and 2 min at 72°C) with 0.1 l of the first-round PCR mixture and primers FP183 (5'-CTTCTGGGCTGTGCTGGTCA-3') and a mixture of unlabeled plus 0.08 pmol of 32 P end-labeled FP184 primer (5'-TCTTTTCTGGGGAT- GTCCAA-3'). A single pair of RNA samples (Fig. 3, Lanes 5 and 6) failed to amplify under these conditions and required 1 l of the first-round PCR mixture. These RT-PCR conditions were found to be within the linear ampli- fication range for RNA isolated from SNB-19 and JEG-3 cell lines (data not shown). UV Cross-Linking and Immunoprecipitation. In preliminary experi- ments, nuclear extracts from SNB-19 and JEG-3 cells were prepared using the small-scale preparation method of Lee et al. (10). However, we then deter- mined that the JEG-3 cell line had high endogenous protease activity and switched to a protocol described by Dyer and Herzog (11). The JEG-3 cells were grown in monolayer culture to 80% confluence for extract preparation. Proteolysis was inhibited by the addition of 5 mM DTT, 1 mM phenylmethyl- sulfonyl fluoride, 10 g/ml leupeptin, 2 g/ml aprotinin, and 1 M pepstatin to the lysis, wash, and extraction buffers. The final protein concentration of the nuclear extracts ranged from 2 to 6 mg/ml. The UV cross-linking experiments were performed using in vitro splicing conditions described previously (12). Capped RNA transcripts were prepared from EcoRI-digested pFGFR-67, -68, or -69 (Fig. 2, WT, M2, and M4, respectively). The 32 P[UTP]-labeled RNA transcripts were incubated with 30% cell nuclear extract, 1% polyethylene glycol, 0.625 mM ATP, 25 mM creatine phosphate, 1 mM MgCl 2 and 20% buffer D at 30°C for 10 min. For competition assays, unlabeled RNA was incubated with the splicing mixture for 10 min on ice before the addition of labeled RNA. After RNase treatment, the UV cross-linked RNA/protein com- plexes were immunoprecipitated with the PTB-specific monoclonal antibody DH3 (a gift from David Helfman, Cold Spring Harbor Laboratories) as described previously (13, 14). Received 10/29/99; accepted 1/17/00. 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 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Public Health Service Grant CA-67946 awarded to G. J. C. by the National Cancer Institute. 2 To whom requests for reprints should be addressed, at Section of Endocrine Neo- plasia and Hormonal Disorders, Box 015, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-2840; Fax: (713) 794-4065; E-mail: gcote@mdanderson.org. 3 The abbreviations used are: FGFR-1, fibroblast growth factor receptor-1; PTB, polypyrimidine tract-binding protein; RT-PCR, reverse transcription-PCR; nt, nucleotide. 1221 Research. on September 19, 2021. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from