A NOVEL NK-2-RELATED TRANSCRIPTION FACTOR ASSOCIATED WITH HUMAN FETALLIVER AND HEPATOCELLULAR-CARCINOMA

A novel cDNA was partially isolated from a HepG2 cell expression libra ry by screening with the promoter-linked coupling element (PCE), a sit e from the alpha-fetoprotein (AFP) gene promoter, The remainder of the cDNA was cloned from fetal liver RNA using random amplification of cD NA ends, The cDNA encodes a 239-amino acid peptide with domains closel y related to the Drosophila factor nk-2. The new factor is the eighth vertebrate factor related to nk-2, hence nkx-2. 8. Northern blot and r everse transcriptase polymerase chain reaction analysis demonstrated m RNA in HepG2, two other AFP-expressing human cell lines, and human fet al liver, Transcripts were not detected in adult liver. Cell-free tran slation produced DNA binding activity that gel shifted a PCE oligonucl eotide. Cotransfection of nkx-2.8 expression and PCE reporter plasmids into HeLa cells demonstrated transcriptional activation; NH2-terminal deletion eliminated this activity. Cotransfection into AFP-producing hepatocytic cells repressed AFP reporter expression, suggesting that e ndogenous activity was already present in these cells. In contrast, co transfection into an AFP-negative hepatocytic line produced moderate a ctivation of the AFP gene, The cardiac developmental factor nkx-2.5 co uld substitute for nkx-2.8 in all transfection assays, whereas another related factor, thyroid transcription factor 1, showed a more limited range of substitution, Although the studies have yet to establish def initively that nkx-2.8 is the AFP gene regulator PCF, the two factors share a common DNA binding site, gel shift behavior, migration on SDS- acrylamide gels, and cellular distribution, Moreover, the nk-2-related genes are developmental regulators, and nkx-2.8 is the first such fac tor associated with liver development.