Characterization of Insulin-Like Growth Factor 1 in Human Primary Brain Tumors1
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(CANCER RESEARCH 5.1. 2475-2478. June I. I993| Advances in Brief Characterization of Insulin-like Growth Factor 1 in Human Primary Brain Tumors1 Ann-Christin Sandberg-Nordqvist,2 Par-Axel Stâhlbom, Manfred Keinecke, V. Peter Collins, Hans von Hoist, and Vicki Sara I)('[nii'tint'iit of Pathology IA-C. S-N.. P-A. S.. V. 5./. Ludwig Institute of Cancer Reseanh ¡V.P. C.I. and Dépariaientof Neurosurgery IH. \: H.¡.Karolinska Hospital. Stockholm. Sweden: BioScience Center ¡P-A.S./. KABI Pharmacia. Stockholm. Sweden: and Institute of Anatomy IM. R.]. University of Zurich. Zurich. Smtyrland Abstract cells, and we have characterized the transcripts for the IGF-I variants found. The complete amino acid sequence of two IGF-I precursor Insulin-like growth factor I (IGF-1) is involved in the regulation of proteins expressed have been deduced from the cDNA sequence iso brain development and has been suggested as an autocrine stimulator of lated from the glioma mRNA. brain tumor cell proliferation. This study demonstrates the expression of K.I -1 in tumor tissue from human gliomas and one esthesioneurohlas- toma. I siim immunohistochemistry. expression of an IGF-1-like peptide was localized in tumor cells of 6 of the 9 gliomas examined as well as the Materials and Methods esthesioneurohlastoma. From one anaplastic oligodendroglioma (which showed strong K.l -I immunostaining) the IGF-I transcripts were char Materials. Tumor material removed at operation was collected and fro/en at -85°C for periods of up to 1 year. The tumors were histopathologically acterized after isolation of mKNA followed by amplification using the reverse transcriptase-polymerase chain reaction. Two IGF-I complemen classified according to the WHO classification and malignancy grading system tary UNAs resulting from alternative splicing of the K.I -1 primary tran (10) with the addition of the criteria of Burger el al. (11) to differentiate script were identified. These transcripts encode two different precursor between anuplastic aslrocytomas and gliohlaslomas. proteins which correspond to Ka IGF-1 and Kb IGF-I. The significance of Immunohistochemistry. The tissue was embedded in paraffin (Histowax). IGF-1 alternative mKNA splicing pathways remains to be determined. Sections of 4 urn were placed on chrome-alun-treated glass slides, dried at 37°Covernight, dewaxed. and rehydrated. Sections were blocked with phos Introduction phate-buffered saline (pH 7.4| containing 2r/r bovine serum albumin and 29r normal goat serum. Primary and secondary antisera were centrifuged at I2.(XH) Gliomas are the most common primary tumors of the central ner rpm tor 15 min before use. A polvclonal antiserum K37 (12) raised in rabbit vous system in adults. At present the prognosis for patients with the was applied at a dilution of 1:2(K) for 12 h at 7°C.After repeated buffer malignant forms of these tumors is poor: however, an understanding washing, biotinylated goat anti-rabbit IgG (1:50; Bioscience Products. Em- of the cellular mechanisms underlying the loss of normal growth menhrucke. Swit/erlandl was applied to the sections. After a second set of control mechanisms in glioma cells may lead to improved therapy. buffer washes, visualization was obtained with streptavidin-fluorescein- Growth factors play an essential role in the regulation of normal isothiocyanate ( 1:30; Bioscience Products). Controls for the specificity of the cellular growth and differentiation and are believed to be involved in reactions were performed both by replacement of the primary antiserum with tumorigenesis. Autocrine, juxtacrine. or paracrine mechanisms may be nonimmune rabbit serum and by preabsorption of the primary antiserum with involved in tumor cell growth. IGF-1' has been implicated as an hlGF-1 (0.4. 4. 40. and 400 ug peplide/ml diluted antiserum). As "positive" autocrine growth stimulator for several tumor types. e.f>.. human controls, sections of rat endocrine pancreas known to contain IGF-1 immu- breast carcinoma cells (1) and small cell lung tumor cells (2). IGF-1 noreactive cells (12) were processed in parallel with each incubation series. is integrally involved in the normal growth and differentiation of the Photographs were taken with a Zeiss Axiophot. nervous system and has been suggested as a putative autocrine stim cDNA Amplification and Sequencing. Tumor tissue from the patient with ulator of tumor growth in the central nervous system (3-5). Expres anaplastic oligodendroglioma was removed from the frontal lobe of the brain and immediati}' placed in liquid nitrogen. Total RNA was isolated by acid sion of IGF-I receptors has been shown in human glioma (3, 6-8). However, it is unclear whether IGF-1 is produced in these tumors or guanidinium thiocyanate-phenol-chloroform extraction (13). Polyadenylated RNA was separated by three cycles of oligodeoxythymidine cellulose chro- what its function would be. Multiple transcripts from the IGF-1 gene are seen in malignant gliomas (5). The IGF-I gene consists of 6 cxons matography (type 3; Collaborative Research). RNA concentration and purity were estimated on the basis of absorhance measurements at 260 and 280 nm. which may be alternatively spliced to result in different transcripts. Five ug polyadenylated RNA were heated at 65°Cfor 5 min and then cooled These include Ea IGF-1 (exons \~^ and 6) and Eb IGF-1 (exons 1-5) on ice. followed by single-stranded cDNA synthesis with an oligodeoxythy (9). The IGF-I transcripts and peptides in gliomas have not been midine primer (cDNA kit; Amersham). One-fifth of the cDNA reaction was characterized. To maintain an autocrine loop hypothesis it is essential amplified by PCR (14) with AmpliTaq DNA polymerase (Perkin Hlmer Cetus) to establish the synthesis of both the growth factor and its receptor by in two steps: first, with primers designed for the 5'-region with EcoR\ sites the tumor cells. The transcripts must be characterized in detail to common to both the IGF-1 mRNAs (Met-1 5'-ATAGAATTC-ATGG- exclude mutations and overexpression. In the present study we dem GAAAAATCAGCAGTCTTCC and Glu-6 .V-ATAGAATTC-CTGGGTCT- onstrate high levels of IGF-1 expression in human malignant glioma TGGGCATGTCGGTGTG). and second, with specific primers with EcuRl sites for the Ea IGF-1 cDNA (Asp-6 5'-ATAGAATTC-GACATGGCCAA- GACCCAGAAG and exon 6 5'-ATAGAATTC-CAGCAATCTACCAACTC- CAGG) and the Eb IGF-1 cDNA (Asp-6 5'-ATAGAATTC-GACATGGCCAA- Received 3/IW93; accepted 4/22/93. The costs of publication of this article were defrayed in pan by the payment of page GACCCAGAAG and exon 5 5'-ATAGAATTC-TTCTTTGCGCTTTCTA- charges. This article must therefore be herehy marked ativi'rtiseinenr in accordance with GGGC) sequences (9). The PCR program was 30 cycles of 94°Cfor I min. 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by the Swedish Medical Research Council, the Swedish Cancer Society, 55°Cfor 2 min, and 72°Cfor 3 min followed by a final extension at 72°Cfor the Cancer Society in Stockholm, the Hans and Loo Osierman Foundation, and the Swiss 5 min. The products were run on a 1% agarose gel to confirm the sizes (Fig. National Foundation. I ). Then the products were reamplified to provide material for subcloning into 2 To whom requests for reprints should be addressed, at Department of Pathology. Karolinska Hospital. Box 6()5(X), S-104 01 Stockholm. Sweden. a pUC vector to the £«>RIsite.The insens were sequenced by the dideoxy 'The abbreviations used are: IGF-1. insulin-like growth factor I; PCR. polymerase chain terminating method using Sequenase version 2.0 (United States Bio chemical Co.) according to the manufacturer's instructions. chain reaction: cDNA. complementary DNA. 2475 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1993 American Association for Cancer Research. IGF-I IN HUMAN PRIMARY BRAIN TUMORS immunoreactivity was found in one case, the presence of IGF-1 im munoreactivity in 2 cases, and no IGF-1 immunoreactivity in one case. A larger number of gliomas should be investigated to determine if there is a correlation between elevated expression of IGF-1 and in creasing malignancy grade. Intense IGF-1 immunostaining was also observed in the single esthesioneuroblastoma examined. In this case the immunoreactivity was similarly distributed in the cells. The IGF-I immunoreactive peptide in the anaplastic oligodendro glioma was characterized. As shown in Fig. 2, the tumor cells in this glioma strongly expressed IGF-I. mRNA was isolated from the tumor tissue and by using PCR two IGF-I cDNAs were identified and their nucleotide sequences determined (Fig. 3). The nucleotide sequences correspond to Ea IGF-1 and Eb IGF-1 cDNA, which arise from the fragment Ea Eb alternative splicing of exon 5 or exon 6 into the IGF-1 transcript (9). Primer o 5 02 0.5 0.2 Ea IGF-1 encodes a precursor protein of 153 amino acids including an (/iM) E peptide of 35 amino acids, and Eb IGF-1 encodes a precursor protein of 195 amino acids containing an E peptide of 77 amino acids. Fig. 1. PCR amplification with specific primers for Ea IGF-1 and Eb IGF-I. Single bands of predicted sizes were confirmed by electrophoresis on 1% agarose gel. Two As shown in Fig. 3, the sequence and length of the E peptides in the concentrations of primers were used (0.5 and 0.2 ^M) to reduce the possibility of non two precursor proteins differ. The amino acid sequence of the mature specific priming. The Ea IGF-I fragment is 300 base pairs long, and the Eb IGF-! IGF-1 protein is identical to that reported in other tissues. This is the fragment is 425 base pairs long. As a marker (M).