Indeed, in a separate experiment transgenic mice treated with the 2 2,4-pyrimidinediamine for 25 days were examined 3 months after cessation of drug administration. together reinforce the pivotal role of EML4-ALK in the pathogenesis of NSCLC in humans, and they provide experimental support for the treatment of this intractable cancer with ALK inhibitors. Keywords:transgenic mouse, surfactant protein C, molecular targeted therapy Lung cancer remains the leading cause of malignancy deaths, with almost 1.3 million people dying annually from this condition worldwide (www.who.int/cancer/en). Although a variety of chemotherapeutic regimens have been developed to treat this intractable disease, their efficacy is limited and depends on malignancy subtype. Non-small-cell lung cancer (NSCLC) accounts for 8085% of all lung cancer cases and is less sensitive to cytotoxic drugs than is small cell lung cancer. Unless tumor cells are surgically resected at an early clinical stage, individuals with NSCLC can expect a median survival time of less than 1 year (1). A subset of individuals with NSCLC (mostly nonsmokers, young females, and SGC2085 those of Asian ethnicity) have been shown to harbor mutations in the epidermal growth factor receptor (EGFR) gene (24). Such mutations result in constitutive activation of the EGFR tyrosine kinase, the oncogenic potential of which has been exhibited in a transgenic mouse system (5). Small-molecule drugs that specifically inhibit the catalytic activity of EGFR have been found to exhibit clinical efficacy in the treatment of NSCLC patients, especially in those with an activated EGFR (6,7). We recently developed a system for the construction of retroviral cDNA libraries from small quantities of clinical specimens (810), and we applied this technology to NSCLC to screen for oncogenes that might be potential drug SGC2085 targets (11). With the use of a focus-formation assay performed with mouse 3T3 fibroblasts, we identified a fusion-type oncogene,EML4-ALK, in an NSCLC specimen of a smoker (12). SGC2085 A small inversion within the short arm of chromosome 2 was found to result in the ligation ofEML4andALK, leading to the production of a fusion protein consisting of the amino-terminal portion of EML4 and the intracellular region of the protein tyrosine kinase ALK. The coiled-coil domain name within this portion of EML4 mediates the constitutive dimerization and activation of EML4-ALK, which is responsible for the generation of transformed cell foci in culture and the formation by these cells of s.c. tumors in nude mice. Although Rabbit polyclonal to AGPAT9 the inv (2)(p21p23) rearrangement responsible for the fusion event occurs recurrently in NSCLC patients, it remains to be demonstrated thatEML4-ALKplays an essential role in the carcinogenesis of NSCLC harboring the fusion gene. To address this issue, we have now designed the expression ofEML4-ALKin lung epithelial cells of transgenic mice. The surfactant protein C gene (SPC) is usually specifically expressed in type 2 alveolar epithelial cells, and a fragment of its promoter has been used widely for establishment of mouse lines that express transgenes specifically in lung epithelial cells (1315). We therefore generated impartial mouse lines in whichEML4-ALKexpression is usually driven by theSPCpromoter, and we found that all such mice develop hundreds of adenocarcinoma nodules in both lungs within only a few weeks after birth. Furthermore, inhibition of EML4-ALK activity with a small-molecule drug induced SGC2085 rapid death of the tumor cells. == Results == == Generation ofEML4-ALKTransgenic Mice. == To generate mice with lung-specific expression ofEML4-ALK, we ligated a fragment of theSPCpromoter (3.7 kbp) to a cDNA for EML4-ALK variant 1 with an amino-terminal FLAG epitope tag (12). The cDNA was, in turn, attached to an RNA splicing cassette and a polyadenylation.