Induction by cycloheximide of the glycoprotein hormone alpha-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 alpha-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 alpha-subunit, including ChaGo (brochogenic carcinoma), FL (amnion), and HeLa (cervical carcinoma). No increase in alpha-subunit mRNA is evident in two choriocarcinoma cell lines (JAr, JEG-3) that produce alpha-subunit as an eutopic product. The half-life of alpha-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 alpha-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 (Mr 50,000) that binds to a DNA fragment located 5' proximal to the alpha-subunit gene but not to more distal 5'-flanking sequence or to the alpha-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 alpha-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 alpha-subunit gene regulation involving a labile repressor and constitute yet another level of differential regulation of the alpha-subunit gene in cells that produce the hormone subunit in an ectopic versus eutopic manner.
Cox
G S
GS
Department of Biochemistry, University of Nebraska Medical Center, Omaha 68198-4525.
Cosgrove
D E
DE
Sullivan
T T
TT
Haas
M J
MJ
D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D005796:Genes / Q000187:drug effects*; D015292:Glycoprotein Hormones, alpha Subunit / Q000096:biosynthesis / Q000235:genetics*; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D011691:Puromycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*
D004268:DNA-Binding Proteins; D015292:Glycoprotein Hormones, alpha Subunit; D012333:RNA, Messenger; D011691:Puromycin; D003513:Cycloheximide