Induction by cycloheximide of the glycoprotein hormone α-subunit gene in human tumor cell lines and identification of a possible negative regulatory factor

The steady-state level of mRNA encoding the glycoprotein hormone α-subunit is increased about 4-fold in HeLa cells by cycloheximide (CHX) or puromycin at concentrations that inhibit protein synthesis. This effect is observed in a number of cell lines that ectopically produce α-subunit, including ChaGo (brochogenic carcinoma), FL (amnion), and HeLa (cervical carcinoma). No increase in α-subunit mRNA is evident in two choriocarcinoma cell lines (JAr, JEG-3) that produce α-subunit as an eutopic product. The half-life of α-subunit mRNA is unchanged in the presence of CHX, but nuclear run-on assays demonstrate a 2.6-fold greater loading of RNA polymerase on the α-subunit gene in nuclei from CHX-treated cells. These results suggest that inhibition of protein synthesis results in higher transcription rates and not in decreased mRNA turnover. A nuclear protein (M(r) 50,000) that binds to a DNA fragment located 5' proximal to the α-subunit gene but not to more distal 5'-flanking sequence or to the α-subunit cDNA has been identified in HeLa but not in JEG-3 cell lines. The p50 DNA binding activity in HeLa cells decreases in the presence of CHX at a rate similar to that at which α-subunit mRNA increases. Moreover, in a series of HeLa cell clones, the levels of p50 are directly proportional to the magnitude of induction produced by CHX. These data are consistent with a model for α-subunit gene regulation involving a labile repressor and constitute yet another level of differential regulation of the α-subunit gene in cells that produce the hormone subunit in an ectopic versus eutopic manner.