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Expression of the transforming growth factor-beta gene during growth inhibition following polyamine depletion
1Department of Surgery, University of Maryland Medical School and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201, USA.
Abstract:
Polyamine depletion and cytokine transforming growth factor-beta (TGF-beta) inhibit cell proliferation. The current study tests the hypothesis that polyamine depletion results in growth inhibition by altering expression of the TGF-beta gene in intestinal epithelial cells. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Cells were grown in DMEM in the presence or absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 6 and 12 days. Administration of DFMO not only depleted intracellular polyamines but also significantly increased the mRNA levels of TGF-beta. Increased TGF-beta mRNA in DFMO-treated cells was paralleled by an increase in TGF-beta content. Depletion of intracellular polyamines by DFMO had no effect on the rate of TGF-beta gene transcription, as measured by nuclear run-on assay. The half-life of mRNA for TGF-beta in normal cells was approximately 65 min and increased to >16 h in cells treated with DFMO for 6 or 12 days. Exogenous polyamine, when given together with DFMO, prevented the increased half-life of TGF-beta mRNA in IEC-6 cells. TGF-beta added to the culture medium significantly decreased the rate of DNA synthesis and final cell number in normal and polyamine-deficient cells. Furthermore, growth inhibition caused by polyamine depletion was partially but significantly blocked by addition of immunoneutralizing anti-TGF-beta antibody. These results indicate that 1) depletion of intracellular polyamines induces the activation of the TGF-beta gene through posttranscriptional regulation and 2) increased expression of the TGF-beta gene plays an important role in the process of growth inhibition following polyamine depletion.
Insights
Polyamine depletion in intestinal cells increases transforming growth factor-beta (TGF-beta) mRNA stability, leading to growth inhibition. This effect is partially reversed by blocking TGF-beta, indicating its crucial role in polyamine-deficient cell growth.
Area of Science:
- Cell Biology
- Molecular Biology
- Gastroenterology
Background:
- Cell proliferation is regulated by polyamines and transforming growth factor-beta (TGF-beta).
- Polyamines are essential for cell growth, and their depletion can inhibit proliferation.
Purpose of the Study:
- To investigate if polyamine depletion inhibits intestinal epithelial cell growth by altering TGF-beta gene expression.
- To elucidate the regulatory mechanisms of TGF-beta gene expression in response to polyamine depletion.
Main Methods:
- Utilized the IEC-6 rat intestinal crypt cell line.
- Administered alpha-difluoromethylornithine (DFMO) to deplete intracellular polyamines.
- Measured TGF-beta mRNA levels, mRNA half-life, gene transcription, and protein content.
- Assessed cell proliferation and DNA synthesis rates.
- Used immunoneutralizing antibodies to block TGF-beta activity.
Main Results:
- DFMO treatment depleted polyamines and significantly increased TGF-beta mRNA levels and content.
- Polyamine depletion did not affect TGF-beta gene transcription rate but increased mRNA half-life.
- Exogenous polyamines prevented the increased TGF-beta mRNA half-life.
- TGF-beta addition decreased DNA synthesis and cell number.
- Anti-TGF-beta antibodies partially blocked growth inhibition caused by polyamine depletion.
Conclusions:
- Polyamine depletion activates the TGF-beta gene through posttranscriptional regulation, specifically by increasing mRNA stability.
- Elevated TGF-beta expression plays a significant role in the growth inhibition observed in polyamine-deficient intestinal epithelial cells.