Thymosin ß-10Gene Overexpression Correlated ...

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Daniela Califano, Carmen Monaco, Giovanni Santelli, Ada Giuliano, Maria Luisa Veronese, Maria Teresa ..... *F. de Nigris, R. Visconti, J. Cerimi, D. Califaro.
[CANCER RESEARCH 58, 823-828.

February 15. 1998]

Thymosin ß-10Gene Overexpression Correlated with the Highly Malignant Neoplastic Phenotype of Transformed Thyroid Cells in Vivo and in Vitro1 Daniela Califano, Carmen Monaco, Giovanni Santelli, Ada Giuliano, Maria Luisa Veronese, Maria Teresa Berlingieri, Vittorio de Franciscis, Nicole Berger, Francesco Trapasso, Massimo Santoro, Giuseppe Viglietto, and Alfredo Fusco2 Servizio di Oncologia Sperimentale "E, " Istituto per lo Studio e la Cura dei Tumori Fondazione "G. Pascale" [D. C.. C. M.. G. S.. A. G., G. V.J. and Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli "Federico li" ¡M.T. B.. V. d. F., M. S.]. 80131 Napoli, Itaiv; Dipartimento dìMedicina Sperimentale e ClÃ-nica,Facoltà di Medicina e Chirurgia di Catanzaro, Università degli Studi dìReggio Calabria. 88100 Catanzaro, Italy [F. T., A. F.]; Kimmel Cancer Center, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [M. L V.I; Laboratoire d'Anatomie Pathologique, Service de Chirurgie, Hôpital de L' Antiquaille, 6921 Lvon Cedex 05, France IN. B.j; and Laboratoire de Cytologie. Hôpital Jules Coumont, 69.ÃŒ10 Pierre Bénite,France [N. B.¡

ABSTRACT A subtractive thyroid cDNA library was constructed from two human thyroid carcinoma cell lines originating from an anaplastic carcinoma and a papillary thyroid carcinoma. The library was used to identify genes correlated with the progression to a highly malignant phenotype. The thymosin ß-10gene was isolated and found to be expressed at much higher levels in the anaplastic cell line than in the papillary cells. The thymosin ß-10gene was overexpressed in five carcinoma cell lines com pared with normal thyroid tissue and normal thyroid primary culture cells. The highest expression occurred in the most malignant cell lines. Thymosin ß-10gene expression was also increased in surgically removed human thyroid carcinomas and was highest in the anaplastic carcinomas. Thymosin ß-10gene expression was correlated with the degree of the malignant phenotype also in rat thyroid cells transfected with cellular and viral oncogenes of different tumorigenicity. These results show that thy mosin ß-10Overexpression is a general event of thyroid cell neoplastic transformation and suggest that the gene is involved in the progression of thyroid carcinogenesis. Finally, the thymosin ß-10gene was located on chromosome

2q37 by fluorescence in situ hybridization

analysis.

INTRODUCTION Thyroid tumors include a wide spectrum of lesions with different phenotypic characteristics and biological behavior: benign adenomas, differentiated carcinomas (papillary and follicular), and undifferentiated or anaplastic carcinomas. A temporal progression from the more benign toward the more aggressive neoplasias has been postulated, and it has been suggested that this process is marked by changes in the expression of different genes (1). Thus, the isolation and character ization of genes differentially expressed by thyroid tumor cells of various degrees of malignancy may shed light on the mechanisms involved in the process of tumoral progression. Subtractive hybrid ization has been successfully used to identify changes in gene expres sion that occur during the genesis and progression of different types of cancer (2). Here we describe the use of a subtractive library screening of two thyroid carcinoma cell lines: ARO, established from a human anaplastic thyroid tumor (3); and NPA, established from a human papillary thyroid tumor (3). The two cell lines have different degrees of malignancy. The ARO cell line grows with a high efficiency in soft agar and induces tumors with a short latency period when injected into athymic mice; conversely, the NPA cell line has a lower growth Received 6/25/97; accepted 11/26/97. 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. 1This work was supported by the Associazione Italiana per la Ricerca sul Cancro, the Consiglio Nazionale delle Ricerche Progetto Finalizzato "Applicazioni Cliniche della Ricerca Oncologica," and thèMinistero della Sanità Fondo Sanitario Nazionale 1994. C. M. and F. T. are recipients of a fellowship from the Associazione Italiana per la Ricerca sul Cancro. A. F. was the recipient of a fellowship from the American-Italian Cancer Foundation. 2 To whom requests for reprints should be addressed, at Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli "Federico II." via Pansini 5, 80131 Napoli, Italy. Phone: (39-81 ) 7463056; Fax: (39-81) 7463037 or 7701016.

efficiency in agar and induces small tumors in athymic mice only after a long latency period (4). The subtractive library screening yielded a differentially expressed cDNA clone, the sequence of which corresponds to that of the human thymosin ß-10cDNA (5). Thymosins were originally isolated from calf thymus, and they might be involved in immunomodulatory func tions (6). The thymosin ßfamily comprises very small (Mr