Identification of differentially expressed nucleolar TGF-beta 1 target (DENTT) in human lung cancer cells that is a new member of the TSPY/SET/NAP-1 superfamily

The transforming growth factor-beta1 (TGF-beta1) responsive epithelial non- small-cell lung cancer (NSCLC) cell line NCI-H727 was used to identify pote ntial target genes involved in TGF-pi-mediated responses. Comparative cDNA expression patterns between cells treated with TGF-beta1 and those treated with vehicle were generated by differential mRNA display, One 496-bp fragme nt, differentially increased threefold by TGF-beta1 and hybridizing to a 2. 7-kb mRNA species in NCI-H727 cells by Northern analysis, revealed no signi ficant match to any known gene sequence, The mRNA transcript of this novel gene that we named differentially expressed nucleolar TGF-beta1 target (DEN TT) is expressed in several normal human tissues, with the highest level of expression in brain. Human brain cDNA library screening and 5 ' rapid ampl ification of cDNA ends yielded fall-length DENTT cDNA containing an 1899-bp open reading frame encoding a predicted 633-amino-acid protein with four p otential nuclear localization signals (NLSs) and two coiled-coil regions. D ENTT contains a conserved 191-residue domain that shows significant identit y to, and defines, the TSPY/TSPY-like/SET/NAP-1 superfamily, Enhanced green fluorescent protein (EGFP)-tagged full-length DENTT transfected into COS-7 cells showed nucleolar and cytoplasmic localization. Transfection of EGFP- tagged DENTT NLS deletion constructs lacking the bipartite NLS-1 were exclu ded from the nucleolus. While NLS-1 is necessary for nucleolar localization of DENTT, it is not sufficient for sole nucleolar localization. Our data s how that DENTT mRNA induction by TGF-beta1 correlates with induction of TGF -beta1 mRNA, induction of extracellular matrix gene expression, and inhibit ion of colony formation in soft agarose in TGF-beta1 responsive NSCLC cells when exposed to TGF-beta1, TOE-pi does not induce DENTT mRNA expression in TGF-beta1 nonresponsive NSCLC cells, Our data suggest that this novel TGF- beta1 target gene has distinct domains for direction to different subnuclea r locations, (C) 2001 Academic Press.