Qualitative and Quantitative Changes of Human Tenascin Expression in Transformed Lung Fibroblast and Lung Tumor Tissues: Comparison with Fibronectin Fumitaka Oyama, Setsuo Hirohashi, Yukio Shimosato, et al. Cancer Res 1991;51:4876-4881. Published online September 1, 1991.
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[CANCER RESEARCH 51, 4876-4881, September 15, 1991]
Qualitative and Quantitative Changes of Human Tenascin Expression in Transformed Lung Fibroblast and Lung Tumor Tissues: Comparison with Fibronectin1 Fumitaka Oyama, Setsuo Hirohashi, Yukio Shimosato, Koiti Titani, and Kiyotoshi Sekiguchi2-3 Institute for Comprehensive Medical Science, Fujita Health University School of Medicine, Toyoake, Aichi 470-11 [F. O., K. T., K. S.J, and Pathology Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104, fS. H., Y. S.] Japan
ABSTRACT Qualitative and quantitative alternations of human tenascin (TN) expression in virally transformed lung fibroblasts and in lung tumor tissues were investigated using S, nuclease protection analysis in com parison with those of fibronectin (FN). Transformed fibroblasts and fetal lung tissues expressed more TN iiiKNA with an extra sequence encoding the sixth FN type HI repeat than did normal cells and adult tissues. The splicing pattern of TN mRNA was also altered in many lung cancer tissues, showing increased or sometimes decreased expression of the TN mRNA with the extra sequence when compared with their surrounding normal tissues. These results provide additional evidence for the oncodevelopmental regulation of alternative RNA splicing in human lung tissues, first observed with FN mRNA (F. Oyama, et al., Cancer Res., 50:1075-1078, 1990). Quantitative analysis of the levels of TN and FN mRNAs showed that the ratio of TN mRNA to FN mRNA was signifi cantly increased in transformed fibroblasts and in some lung tumor tissues, when compared with their normal counterparts. Among different types of lung tumors, a significant increase of the TN/FN ratio was observed with most squamous cell carcinoma but with only a small fraction of adenocarcinoma. Since TN has been shown to inhibit cell adhesion to FN, the altered ratio of TN mRNA to FN mRNA may well affect the adhesive and migratory properties of tumor cells in lung cancer tissues.
INTRODUCTION TN4 is a novel extracellular
matrix glycoprotein with tem
porally and spatially restricted tissue distribution (1, 2). TN is transiently expressed in many developing organs such as con nective tissues, the mesenchyme of epithelial organs, and the central and peripheral nervous system (3-7). Although expres sion of TN is suppressed in most adult tissues, TN reappears in the stroma of tumors and healing wounds (5, 7, 8). TN exists as a hexamer of M, 220,000-320,000 subunits which are linked by disulfide bonds near the /V-terminus. Recent studies on sequencing of TN cDN As revealed that TN consists of three types of structural modules, i.e., epidermal growth factor-like repeat, FN type III repeat, and a domain with a sequence homology to ßand 7-chains of fibrinogen (Refs. 911; see also Fig. \A). The TV-terminal region of TN contains four heptad repeats composed of hydrophobic amino acid resi dues possibly involved in the trimer formation, followed by 13 epidermal growth factor-like repeats. The central region is made up of 8 to 15 FN type III repeats, the number of which varies Received 6/12/91; accepted 7/8/91. 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. ' This work was supported by Grants-in-Aid from the Ministry of Education. Science, and Culture of Japan; a Special Coordination Fund from Science and Technology Agency of Japan; and grants from Fujita Health University and the Naito Foundation. 2 Present Address: Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo, Izumi, Osaka 590-02, Japan. 5To whom requests for reprints should be addressed. * The abbreviations used are: TN, tenascin; FN, fibronectin; PCR, polymerase chain reaction; cDNA, complementary DNA.
depending on the species and the cell type. The domain ho mologous to fibrinogen is located at the C-terminal region. TNs isolated from various sources often display a character istic pattern of a large and two small subunit forms upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12). Recent studies have shown that TN polymorphism is generated by alternative splicing of a single pre-mRNA which results in exclusion of up to 7 FN type III repeats present in the middle of the subunit polypeptides (10, 11, 13). The regulation of the alternative splicing of TN pre-mRNA in vivo and in vitro has been only poorly understood. FN is another extracellular matrix glycoprotein which dis plays complex molecular heterogeneity due to alternative RNA splicing (14-16). FN is assumed to play an important role in many aspects of cell-substrate interactions including cell adhe sion, migration, differentiation, and malignant transformation (14). Recently, we found that the alternative splicing of FN premRNA at extra type III regions termed ED-A and ED-B was oncodevelopmentally regulated in human liver and lung tissues (17,18). Normal liver exclusively expressed the ED-A" mRNA, whereas fetal and cancerous liver tissues expressed a significant amount of ED-A+ mRNA (17). Similarly, an increased expres sion of the ED-B+, but not ED-A*, mRNA was observed in fetal and cancerous lung tissues (18). TN and FN show a great deal of structural similarities. More than half of these proteins are built up from FN type III repeats. Both proteins display molecular polymorphism due to the alternative RNA splicing occurring at the regions encoding FN type III repeats. Despite these structural similarities, these two proteins significantly differ in their biological properties. Al though TN contains a cell-adhesive Arg-Gly-Asp (RGD) se quence, it has a very weak activity, if any, to mediate cell attachment and has been reported to inhibit cell adhesion to the FN-coated substrate (12,19). The physiological significance of the similarities and differences between TN and FN has not yet been fully addressed. In the present study, we investigated the patterns of alterna tive RNA splicing and levels of transcripts of human TN in cultured lung fibroblasts and in normal, fetal, and cancerous lung tissues in order to gain an insight to regulation of TN expression in vivo and in vitro. Our results showed that the alternative splicing of TN pre-mRNA was indeed altered in virally transformed fibroblasts as well as in fetal and cancerous lung tissues, and the ratio of TN mRNA to FN mRNA was significantly altered in transformed cells and certain types of lung cancer tissues. MATERIALS
AND METHODS
Materials. S, nuclease was purchased from Boehringer Mannheim Yamanouchi (Tokyo, Japan). Klenow fragment of DNA polymerase I, T4 polynucleotide kinase, EcoT14I, ///ndlll, Smal, and pUCl 19 were from Takara Shuzo (Kyoto, Japan). [
