Identification of a Series of Transforming Growth Factor β-Responsive Genes by Retrovirus-Mediated Gene Trap Screening

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Abstract
Transforming growth factor β (TGF-β) plays important roles in the regulation of proliferation, differentiation, apoptosis, and carcinogenesis. To identify genes responsible for maintaining the phenotype induced by TGF-β, we performed a retrovirus-mediated gene trap screening designed to isolate TGF-β-responsive genes in human lung carcinoma cell line A549. After screening 249 trap lines, 21 were found to express the reporter β-galactosidase gene in a TGF-β-responsive manner. Interestingly, in large proportions of these trap lines, the reporter gene was responsive also to phorbol ester and was suppressed by gamma interferon. Fragments of all these trapped genes were recovered by 5′- and 3′-rapid amplification of cDNA ends (RACE), and in 15 out of 21 cases (71%), the TGF-β responsiveness of the endogenous genes was confirmed by RNA blot hybridization. In at least five cases, the TGF-β-induced upregulation was found to be cycloheximide resistant, suggesting the roles of the genes in the TGF-β-induced primary responses. Sequence analyses revealed that 43% (9 of 21) of the trapped genes were novel and that the remainder included genes previously reported to be upregulated by TGF-β, such as epidermal growth factor receptor and β1 integrin, documenting the validity of this approach. Other known genes include the ones encoding the proteins associated with cell proliferation (ribosomal proteins S15a, hNRP/NAP-1, and lipocortin II), focal adhesions (paxillin), and transcriptional regulation (thyroid hormone receptor activator molecule 1 [TRAM-1]).