Abstract
The coordinate regulation of human chorionic gonadotropin (hCG) subunit synthesis by JEG-3 choriocarcinoma cells was studied at the pretranslational level. The responses of the hCG alpha and beta mRNAs were measured during stimulation with the potent cAMP analog 8-bromo-cAMP (8-Br-cAMP) using 32P-labeled hCG alpha and beta cDNA probes. The hCG alpha mRNA (850 bases) and beta mRNA (1050 bases) from JEG-3 cells were identical in size to that of their respective mRNAs from placenta, by Northern blot analysis. After 48 h of stimulation with 2 mM 8-Br-cAMP, production of immunoreactive alpha and beta subunits increased 25- and 52-fold, respectively; corresponding levels of the alpha and beta mRNAs increased 36- and 43-fold, respectively, in a dot blot hybridization assay. Total cellular protein, DNA content, and messenger RNA pools were not altered by treatment with 8-Br-cAMP. The temporal coordination of the expression of the hCG alpha- and beta-subunit genes was examined by comparing the time course of stimulation of the respective mRNAs and the production of immunoreactive subunits. The kinetic responses of the alpha and beta mRNAs differed: the increase in hCG alpha mRNA preceded the increase in hCG beta mRNA, while levels of free alpha subunit and intact hCG increased in parallel with the increase in beta mRNA. hCG alpha mRNA levels increased rapidly between 8 and 24 h after the addition of 8-Br-cAMP, and approached a plateau by 48 h. The levels of hCG beta mRNA increased steadily throughout the 8-48 h period. These results demonstrate that the cAMP analog 8-Br-cAMP differentially regulates hCG subunit biosynthesis in JEG-3 cells at a pretranslational level, and that the stimulation by 8-Br-cAMP in this system appears to be relatively selective for hCG subunits.
Highlights
The coordinate regulation of human chorionic go- sequence of the a subunit of all four hormones is identical nadotropin subunit synthesis by JEG-3 chorio- within a given species, but the differing,!3 subunits confer carcinoma cells wasstudied at thepretranslational biologic and immunologic specificity to each hormone [1]
DNA content, and messenger RNA pools were not al- found in excess [5], suggesting that thetwo subunits may be tered by treatment with8-Br-CAMP
The temporal coordination of the expression of the hormone subunit synthesis hasbeen observed for thyrotropin hCG a-and &subunit genes was examined by compar- [6,7,8] and lutropin [8, 9], and it has been proposed that the ing the time course of stimulation of the respective control of glycoprotein hormone expression resides in the
Summary
Little is known about the physiological regulators of hCG in uiuo, hCG production by placental explants is stimulated by cAMP analogs (lo), suggestmRNA increased steadily throughout the 8-48 h pe- ing that cAMP may play a role in thephysiologic modulation riod These results demonstrate that thecAMP analog 8- tion by neoplastic trophoblastic cell lines (10, l l ) , and these. Br-CAMP differentially regulateshCG subunit biosyn- cell lines have become useful models for studying regulation thesis in JEG-3 cells at a pretranslational level, and of hCG production. We have examined the mechanism of cAMP stimulation more directly, by using cDNA probes to measure levels of the specific mRNAs for the hCG a and p subunits in JEG-3. Sulfate, 50% formamide, and 300pg/ml denatured salmon sperm DNA
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