Glucocorticoids rapidly inhibit the expression of c-myc mRNA in P1798 lymphoma cells. Statistically significant decreases can be observed within 5-10 min after the addition of glucocorticoids. Although transcription of c-myc decreases within a few hours after dexamethasone is added to P1798 cell cultures, nuclear run-on transcription cannot be used to demonstrate that the very early changes in mRNA abundance reflect corresponding changes in transcriptional activity. An RNase protection assay has been used to measure the abundance and rates of turnover of the two major c-myc transcripts arising from the P1 and P2 initiation sites. The relative rates of synthesis of the c-myc mRNAs (i.e. transcription) can be calculated from such data. The abundance of the P2 transcript exceeds that of P1 mRNA by 3- to 4-fold in midlog phase cells. The turnover rates of the two c-myc mRNAs are essentially identical (0.02 min-1), indicating that the P2 promoter is 3-4 times stronger than P1. This was confirmed by measuring the relative transcriptional activities of templates containing the individual c-myc promoters in P1798 extracts in vitro. The expression of P1 and P2 mRNAs decreases at different rates in glucocorticoid-treated cells. A 50% decrease in the abundance of P1 mRNA occurs within 1 h after the addition of dexamethasone. Expression of P2 mRNA is reduced by 50% within 4 h. However, the turnover rates of the major c-myc transcripts do not change in glucocorticoid-treated cells. The t1/2 values of P1 and P2 mRNAs are about 25-30 min and not different from the turnover rates measured in control cells. The data indicate that glucocorticoid regulation of the expression of c-myc in P1798 cells is almost entirely mediated through changes in transcription. Furthermore, glucocorticoids influence c-myc promoter utilization. The P1 promoter is inhibited to a greater extend, although utilization of the P2 promoter is reduced by more than 75%. As a consequence, the P2 promoter is 16-20 times stronger than P1 in glucocorticoid-treated P1798 cells. The physiological significance of this shift in promoter utilization is unknown, but the data suggest that different DNA-protein and/or protein-protein interactions may govern the strength of the two major c-myc promoters.
ASJC Scopus subject areas
- Molecular Biology