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Glucocorticoid regulation of c-myc promoter utilization in P1798 T-lymphoma cells
T Ma1, P B Mahajan, E A Thompson
1Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Abstract:
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Glucocorticoids quickly reduce c-myc mRNA levels in lymphoma cells by affecting transcription, not mRNA turnover. This rapid inhibition highlights glucocorticoid action on gene expression regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Glucocorticoids are potent regulators of gene expression with known effects on cell proliferation and differentiation.
- The c-myc gene is a critical proto-oncogene involved in cell growth and is often dysregulated in cancer.
- Understanding the precise mechanisms by which glucocorticoids regulate c-myc expression is crucial for cancer therapy.
Purpose of the Study:
- To investigate the rapid effects of glucocorticoids on c-myc mRNA expression in P1798 lymphoma cells.
- To differentiate between transcriptional regulation and post-transcriptional mechanisms (mRNA turnover) in glucocorticoid-induced c-myc inhibition.
- To compare the regulation of the two major c-myc transcripts (P1 and P2) under glucocorticoid treatment.
Main Methods:
- Utilized RNase protection assays to quantify c-myc mRNA abundance and turnover rates.
- Employed nuclear run-on transcription assays to assess transcriptional activity.
- Performed in vitro transcription assays using P1 and P2 promoter templates.
Main Results:
- Glucocorticoids rapidly decrease c-myc mRNA abundance within 5-10 minutes in P1798 cells.
- While overall transcription decreases, early mRNA changes are not solely explained by transcriptional inhibition.
- P2 c-myc mRNA is 3-4 times more abundant than P1 mRNA due to a stronger P2 promoter, with similar turnover rates for both.
- Glucocorticoid treatment differentially affects P1 and P2 mRNA expression rates, with P1 decreasing faster than P2.
- mRNA turnover rates remain unchanged, indicating regulation occurs at the transcriptional or post-transcriptional processing level, not degradation.
Conclusions:
- Glucocorticoids rapidly inhibit c-myc mRNA expression in lymphoma cells primarily through transcriptional regulation, not by altering mRNA stability.
- The differential regulation of P1 and P2 c-myc transcripts suggests complex control mechanisms.
- These findings provide insights into the molecular basis of glucocorticoid therapy in lymphoma and other cancers.