Murine lung epithelial (MLE) cell lines representing the distal bronchiolar and alveolar epithelium were produced from lung tumors generated in transgenic mice harboring the viral oncogene simian virus 40 (SV40) large tumor antigen under transcriptional control of a promoter region from the human surfactant protein C (SP-C) gene. The cell lines exhibited rapid growth, lack ofcontact inhibition, and an epithelial cel morphology for 30-40 passages in culture. MicroviUi, cytoplasmic multivesicular bodies, and multilamellar inclusion bodies (morphologic characteristics of alveolar type II cells) were detected in some of the MLE cell lines by electron microscopic analysis. The MLE cells also maintained functional characteristics of distal respiratory epithelial cells including the expression ofsurfactant proteins and mRNAs and the ability to secrete phospholipids. Expression of the exogenous SV40 large tumor antigen gene was detected in al of the generated cell lines. The SP-C/SV40 large tumor antigen transgenic mice and the MLE cell lines will be useful for the study of pulmonary surfactant production and regulation as wel as lung development and tumorigenesis. (4), and synthesis of SPs (5) in culture.Clara cells and type II epithelial cells also serve as progenitor cells of the distal respiratory epithelium in the adult lung (6, 7) and are believed to be the cell of origin forThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. pulmonary adenocarcinomas, a subtype of non-small-cell lung cancer (8). We have previously produced transgenic mice harboring the simian virus 40 (SV40) large tumor antigen (TAg) under the transcriptional control of regulatory sequences derived from the human SP-C promoter region to study the development of pulmonary adenocarcinomas in vivo (9). Transgenic mice bearing the exogenous SP-C/TAg chimeric gene developed pulmonary tumors within 4-6 months of age. The presence ofmicrovilli and lamellar bodies by electron microscopy, as well as the presence of respiratory epithelial cell markers by in situ hybridization, were consistent with the identification of the tumor cells as both bronchiolar and alveolar subtypes in vivo. While cells of the proximal respiratory epithelium and lung epithelial cells of fetal origin (10) have been established in culture, distal respiratory epithelial cell lines that maintain a differentiated phenotype in culture have not been previously produced. In the current study, we describe the production and characterization of distal respiratory epithelial cell lines derived from the SP-C/TAg transgenic mice.

ABS1hACT Continuous cell lines that secrete both insulin and somatostatin were established by two cooprating groups of investigators from a serially transplantable, radiation-induced, rat islet cell tumor. The cell lines, named RIN-r and RIN-m, were initiated from tumors maintained in inbred rats or in athymic nude mice, respectively. The cultured cells are ppithelioid, free of fibroblast.contamination, and can be cloned.They have a hypodiploid chromosome number, are tumorigenic, and possess amine-handling properties, including high evels of L-dopa decarboxylase and formaldehyde-induced fluorescence. Preliminary analysis of clones revealed a spectrum of insulin secretion from undetectable to relatively high. These clonal cell lines provide important systems to study the biology of insulin and somatostatin. MATERIALS AND METHODS Origin of the cultures. Cultures were initiated from a transplantable islet cell tumor (6) induced by high-dose x-irradiation in an inbred NEDH (New England Deaconess Hospital) rat. The tumor was maintained by serial transplantation in NEDH rats. After nine transplants, it was successfully heterotransplanted into athymic nude mice, BALB/c background (ARS/Sprague-Dawley, Madison, WI). Continuous cell lines were derived either from rat transplants (Joslin group) or from nude mouse heterotransplants (NCI-VA group). These cell lines were named RIN-r and RIN-m, respectively.Establishment of RIN-r Cell Line. Tumors were removed aseptically from recipient rats and cut into small fragments The tumor cells were separated from the connective tissue stroma by washing the fragments with tissue culture medium 199 containing 0.1% fetal bovine serum, 16.5 mM glucose, and 400 units of penicillin per ml. The culture medium used throughout the remainder of the procedure was similar, but contained 10% fetal bovine serum. Erythrocytes present in the original cell suspension were removed by centrifugation (750 X g, 10 min) on a discontinuous gradient consisting of a solution of 25% di-

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This study suggests that inactivation of the CDKN2 gene is an essential step in the etiology of malignant mesotheliomas. Defining the role of the p16INK4:Rb tumor suppressor pathway and its immediate downstream substrates will be an important goal in designing future therapeutic strategies.